Devices and methods for reducing matrix effects

ABSTRACT

Devices and methods are provided for reducing matrix effects in protein precipitated bioanalytical samples comprising: a support, and a sorbent associated with the support capable of binding matrix interfering agents present in the bioanalytical sample, wherein the device further comprises filtering means for removing precipitated protein particles. The filtering means is a size exclusion filter or a polymeric or inorganic monolith having a maximum pore size less than or equal to the diameter of the particles to be removed from the sample, and can be integral with the sorbent or associated with the sorbent. The sorbent is characterized by sufficient selectivity between the matrix interfering agents and analytes of interest to provide retention of the matrix interfering agents while providing elution of the analytes of interest (e.g., a reversed phase or a polar modified reversed phase). Typical devices incorporating these features include luer syringe filters, individual filter cartridges, multiwell plates, pipette tips, or inline columns for multiple or single use.

FIELD OF THE INVENTION

This invention relates generally to devices and methods for preparingbioanalytical samples for analytical testing.

BACKGROUND OF THE INVENTION

Bioanalytical testing and quantitation methods suffer from interferencefrom contaminants that can decrease or increase sensitivity to variousanalytes disproportionately to their abundance in the sample. Liquidchromatography-mass spectrometry/mass spectrometry

(LC/MS-MS) is the preferred method for drug metabolism studies; howevermatrix effects can lead to significant analytical errors and should beinvestigated to ensure that precision, selectivity and sensitivity arenot compromised. (Little, J. L. et al. (2006) J. Chromatog. 833, 219).In particular, phospholipids such as phosphatidylcholines interfere withanalyte ionization in electrospray MS detection by reducing analytesensitivity, commonly referred to as ion suppression or matrix effects.See Ahnoff, M. and Hagelin, H. “Matrix Effects in ElectrosprayIonization: Characterization of Plasma Phospholipids asSuppressors/Enhancers of Ionization Efficiency,” presented at theAmerican Society for Mass Spectrometry, 52^(nd) Conference on MassSpectrometry (2004)). Further compounding the problem is that differinglipid composition of different samples, such as blood plasmas fromvarying animal species, can change the response of the analyte and causeproblems in quantitation. These matrix effects are further discussed bythe following presentations by Bennett, et al., which report thedivergent calibration curves and the retention time shifts that canresult from phospholipid contamination of bioanalytical samples:“Managing Phospholipid-Based Matrix Effects in Bioanalysis,”www.tandemlabs.com/capabilities_publications.html (accessed Feb. 26,2007); “A Source of Imprecision Resulting from Ionization Suppressionfrom Strongly Retained Phospholipids and Dioctyl Phthalate,” presentedat the American Society for Mass Spectrometry, 52^(nd) Conference onMass Spectrometry (2004)).

In addition, the presence of contaminants can result in incompletesolvent extraction and hence underreporting of analyte concentrations,or can build up on analytical instrumentation, destroying sensitivity orresulting in downtime while cleaning procedures are instituted.Contaminants such as phospholipids have a tendency to build up on atypical reverse phase HPLC columns during repeated analyses ofprecipitated plasma samples. Accumulated phospholipids can bleed off insubsequent injections, causing a drift in analyte sensitivity over thecourse of multiple injections. See Bennett and Liang, “Overcoming MatrixEffects Resulting from Biological Phospholipids Through SelectiveExtractions in Quantitative LC/MS/MS,” presented at the American Societyfor Mass Spectrometry, 52^(nd) Conference on Mass Spectrometry (2004).Removing the phospholipids requires extensive solvent washing toregenerate a column to proper condition.

Various approaches have been utilized in an attempt to solve theseproblems. Current methods for the removal of phospholipids frombioanalytical samples including liquid/liquid extraction (LLE) and solidphase extraction (SPE) are complicated and require a good deal of methoddevelopment and the potential for analyte losses. For example, use ofstronger eluting strength solvents in SPE can paradoxically result indecreased sample detection due to matrix effects, presumably due tocontamination of the sample with phospholipids. However, limiting theeluting solvent strength to avoid contamination of samples withphospholipids can result in incomplete recovery of less polar analytes.LLE approaches require excessive amounts of labor and time to removecontaminating phospholipids, such as performing extraction andseparation steps, and drying down or freezing samples, in order toremove the contaminants. For example, Bonfiglio et al. discuss theability of several common extraction procedures to remove endogenousplasma components that cause ion suppression in electrospray ionizationtandem mass spectrometry. LLE using methyl-t-butyl ether, SPE with Oasisand Empore, and acetonitrile (ACN) protein precipitation samplepreparation methods were compared. These researchers found that ACNprotein precipitated samples showed the greatest amount of ionsuppression while LLE extracts demonstrated the least. In addition, theion suppression was found to be analyte dependent, and associated withthe most polar analyte. The least ion suppression for all analytes wasobserved in samples treated with both LLE and SPE. The authors concludethat there were most likely multiple endogenous components involved inion suppression, and that the effects may persist well after theinjection into the HPLC system is made, resulting in the collection ofinvalid data. A further filtration step was suggested in an effort toprovide yet cleaner samples for analysis. (Bonfiglio, R., et al. (1999)Rapid Comm. Mass. Spectr. 13, 1175). However, use of multiple samplepreparation steps is labor and time intensive, and increases the cost ofperforming analyses.

More recently, new approaches for removing phospholipids from sampleshave been attempted. For example, Johanson reported that use of strongcation exchange column to remove cationic lipids includingphosphatidylcholines from lipid extracts resulted in the ready detectionof peaks that had been completely suppressed in the crude extract.(Johanson, R. A., et al. (2007) Anal. Biochem. 362, 155). U.S. PatentApplication Publication No. 20050054077 (Bennett, et al.) describesdevices and methods for removing phospholipids from biological samplesinvolving the use of a phospholipotropic multivalent cation coupled to asupport. Such cations reportedly include transition metals, lanthanidesor actinides, preferably cerium. Use of these phospholipotropicmultivalent cation sorbents was further described by Van Home, et al.,describing the sorbents as possessing high oxophilicity for thephosphate groups on the phospholipid molecules. (Van Home, K. C., et al.“Preventing Matrix Effects By Using New Sorbents to Remove Phospholipidsfrom Biological Samples” (2003) presented at the Proceedings of theAmerican Association of Pharmaceutical Scientists Conference). Theauthors reported a goal of providing facile removal of phospholipidsfrom biological samples and extracts utilizing an extraction chemistrythat would not remove desirable pharmaceutical analytes. Many differentmechanisms reportedly were evaluated, including reverse-phase (nonpolar)and both anion and cation exchange. Phospholipid extraction wasimplemented via extraction sorbents used alone or in combination withprotein precipitation, liquid liquid extraction (LLE) or solid phaseextraction (SPE). In particular, the phospholipid content of extractsreconstituted in methanol was reportedly reduced by as much as 94-96%using the lanthanide sorbent alone. Use of a lanthanide extractionsorbent to remove phospholipid from a protein precipitated samplereconstituted in methanol reportedly resulted in phospholipid removal ofabout 92%-98% and enhanced detection of spiked analytes. However, theprocedure required centrifugation followed by solvent evaporation andreconstitution, which is labor and time consuming and adds to the costsof performing analysis. Use of a lanthanide extraction sorbent to removephospholipid from a sample after methyl-t-butyl ether LLE reportedlyresulted in phospholipid removal of about 95%-97% but withoutsignificant enhanced detection of spiked analytes. The authors concludedthat immobilized lanthanide metal centers are an essential element forhighly selective binding for phospholipid extraction via binding to thephosphate groups.

Shen, et al. describe an evaluation of three different types ofion-exchange solid phase extraction media in an effort to determine theabilities of the media to remove phospholipids from analyte solutions.These authors reported that mixed mode phases fulfill the requirement ofretaining both analytes and diverse metabolites, while reverse-phaseretention mechanisms were detrimental in eliminating ion suppressioncaused by late eluting phospholipids, and advised using an ion exchangemechanism alone rather than mixed mode extraction phases. (Shen, J. X.,et al. (2005) J. Pharm. Biomed. Anal. 37, 359).

U.S. Pat. No. 5,885,921 to Krupey describes the use of hydrophobicsilica adsorbents for the removal of lipids in samples, sold under thebrand name Cleanascite™ for lipid adsorbent and clarification agent forpretreatment of samples prior to further purification. The adsorbent isadded to samples and then the sample is centrifuged to remove theadsorbent containing bound impurities. However, this procedure requirestwo steps to add the sorbent and then remove it, and risks removal ofanalytes from samples.

U.S. Pat. No. 5,759,549 to Hiltunen describes the use of supercriticalfluid extraction for the isolation of lipids from mixtures of lipids.However, this procedure requires specialized equipment and adds to theexpense and cost of removing lipids from samples.

Little et al. describe an “in source multiple reaction monitoring”method for monitoring method development of pharmaceutical analysis inan effort to determine whether there are co-eluting matrix constituentsresulting in ion suppression of analytes of interest. However, theseprocedures do not remove the matrix constituents causing the ionsuppression, but merely attempt to work around the problem, and requirethe elution of the most hydrophobic lipids from the column after eachanalysis. (Little, J. L. et al. (2006) J. Chromatog. 833, 219).

Therefore, numerous methods for removing lipid contaminants inbiological samples are available, although the procedures are time andlabor intensive. Further, samples also contain contaminating proteins,which must also be removed. However, use of denaturing solvents toeffect precipitation results in greater extraction of lipid contaminantsand significant ion suppression of analytes present. Therefore, there isa minimum of two steps needed to prepare a bioanalytical sample foranalysis, if the researcher hopes to maintain his equipment in goodworking order, and to achieve reliable and accurate quantitation ofbioanalytes. When working with small sample volumes, or when multipletesting is needed, such multiple sample preparation procedures arelikely to reduce sample such that an insufficient amount remains for thetesting required. In addition, multiple sample preparation steps resultin a loss of researcher time and labor and raise the costs for theanalytical testing laboratory.

Accordingly, there remains a need for rapid procedures that can removeboth phospholipids and other agents causing matrix effects and proteinsfrom a bioanalytical sample prior to performance of analyticalprocedures.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the invention to provideprocedures for reducing matrix effects in bioanalytical samples.

It is a further object of the invention to provide procedures that canbe performed rapidly to remove phospholipids, surfactants, and proteinsfrom a bioanalytical sample prior to performance of analyticalprocedures.

It is yet a further object of the invention to provide procedures forreducing matrix effects that can be performed in the ordinary course ofsample preparations and that do not require additional steps orexpensive equipment.

Accordingly, there is provided a device for reducing matrix effects in aprotein precipitated bioanalytical sample comprising: a support, and asorbent associated with the support capable of binding matrixinterfering agents present in the bioanalytical sample, wherein thedevice further comprises filtering means for removing precipitatedprotein particles. The filtering means is characterized in having poresizes between about 0.05 μm and about 0.5 μm in diameter for removingprecipitated protein particles present in the sample. In preferredembodiments, the filter means has pore sizes between about 0.1 μm andabout 0.2 μm in diameter, and in particular embodiments, the filtermeans comprises pores of 0.2 and 0.45 μm in diameter. Preferably, thefiltering means is selected from a size exclusion filter or a polymericor inorganic monolith having a maximum pore size less than or equal tothe diameter of the particles to be removed from the sample, and can beintegral with the sorbent or associated with the sorbent. Preferably,when the filtering means is integral with the sorbent, the filteringmeans is a porous inorganic monolith having macropores of a diametersufficiently small so as to exclude particles from the sample, and thesorbent is a reversed phase or polar modified reversed phase bonded tothe porous inorganic monolith. Preferably, the filtering means iseffective to provide optical clarity to the protein precipitatedbioanalytical sample (e.g., % T at 524 nm>95%).

The sorbent is characterized by sufficient selectivity between thematrix interfering agents and analytes of interest to provide retentionof the matrix interfering agents while providing elution of the analytesof interest. Preferably, the sorbent is characterized by a selectivitygreater than 1 for matrix interfering agents and analytes of interest,and in certain embodiments, the selectivity is at least 1.1, inadditional embodiments, the selectivity is at least 1.2, in yet otherembodiments, the selectivity is at least 1.3, in additional embodiments,the selectivity is at least 1.4, and in certain especially preferredembodiments, the selectivity is at least 1.5. Preferably, the sorbentcomprises a reversed phase or a polar modified reversed phase, and inparticular embodiments, the polar modified reversed phase is an amidemodified reversed phase. The sorbent selectivity for matrix interferingagents and analytes of interest is such that when the bioanalyticalsample comprises at least 50% (v/v) denaturing organic solvent, thesorbent retains matrix interfering agents while not retaining analytesof interest. Preferably, the sorbent binds at least 50% of the matrixinterfering agents present in the bioanalytical sample, while providingrecovery of at least 75% of the analytes in the solvent output from thedevice, and more preferably, the sorbent provides for recovery of atleast 90% of the analytes. In preferred embodiments, the sorbent bindsat least 70%, or more preferably at least 85%, or more preferably atleast 90%, and even more preferably at least 95%, and most preferably atleast 99% of the matrix interfering agents.

Typical matrix interfering agents include surfactants, lipids,excipients, or dosing agents. Typical implementations of the device areadapted for use as luer syringe filters, individual filter cartridges,multiwell plates, pipette tips, or an inline columns for multiple orsingle use. In additional embodiments, the support further comprisesreservoir means for performing protein precipitation within the device.

The invention further provides methods for preparing a sample comprisingmatrix interfering agents and proteins for analysis. Typical analysesinclude chromatographic, spectrophotometric, mass spectrometric, and thelike, and combinations thereof. For example, an exemplary analysismethod in the bioanalytical arts for determining pharmaceutical analytesis LC/MS-MS. Accordingly, there are provided methods for reducing matrixeffects and removing protein precipitates in a bioanalytical sample,said methods comprising: a) providing a device comprising a support, anda sorbent associated with the support, wherein said sorbent ischaracterized by a selectivity greater than 1 for matrix interferingagents relative to analytes of interest present in the bioanalyticalsample, and further comprising filtering means for removing proteinprecipitates present in the sample; b) contacting the bioanalyticalsample with the sorbent; and c) eluting the analytes from the sorbentwhile retaining the matrix interfering agents and precipitated proteins,wherein the amount of matrix interfering agents and proteins in theresulting treated sample is reduced. In certain embodiments, the methodfurther comprises precipitating the proteins in the bioanalytical samplein the device prior to or simultaneously with the step of contacting thebioanalytical sample with the sorbent. Preferably, step c) is performedusing vacuum, centrifugal force or positive pressure to cause the sampleto pass through the sorbent and the filtering means, thereby removingmatrix interfering agents and precipitated proteins. Preferably, thefiltering means is characterized in having pore sizes between about 0.05μm and about 0.5 μm in diameter for removing precipitated proteinparticles present in the sample, and in certain embodiments, thefiltering means comprises pore sizes between about 0.1 μm and about 0.2μm. In particular embodiments, the filtering means comprises pores sizesof 0.1, 0.2 and 0.45 μm. Preferably, the matrix interfering agents aresurfactants, lipids, excipients, or dosing agents, and in preferredembodiments, the lipids are phospholipids, and the surfactants areselected from anionic surfactants or nonionic surfactants. Preferablythe surfactants comprise a hydrocarbon chain which can be advantageouslyretained using the sorbents described herein. Preferably, the sorbent ischaracterized by sufficient selectivity between the matrix interferingagents and analytes of interest to provide retention of the matrixinterfering agents while providing elution of the analytes of interest.In certain embodiments, the sorbent comprises a reversed phase or apolar modified reversed phase. In particular embodiments, there areprovided methods for reducing matrix effects and removing precipitatedproteins in a protein precipitated bioanalytical sample comprisingmatrix interfering agents and analytes of interest, the methodcomprising passing the sample through the devices described herein.

In additional embodiments, methods are provided for reducing matrixeffects in a protein precipitated bioanalytical sample comprising matrixinterfering agents and analytes of interest, the method comprising: a)providing a device comprising a support, and a sorbent associated withthe support, wherein said sorbent is characterized by a selectivitygreater than 1 for matrix interfering agents relative to analytes ofinterest present in the bioanalytical sample; b) contacting thebioanalytical sample with the sorbent; and c) eluting the analytes fromthe sorbent while retaining the matrix interfering agents, wherein theamount of matrix interfering agents in the resulting treated sample isreduced. The device can further comprise filtering means for removingprecipitated protein particles. In preferred embodiments, when thebioanalytical sample comprises at least 50% (v/v) denaturing organicsolvent, the sorbent retains matrix interfering agents while notretaining analytes of interest. In additional embodiments, the sorbentretains matrix interfering agents while not retaining analytes ofinterest even at 66%, 75%, or even 90% (v/v) organic solvent, or in thepresence of pH modifiers (e.g., acids, bases). Preferably, the sorbentbinds at least 50% of the matrix interfering agents present in thebioanalytical sample, while providing recovery of at least 90% of theanalytes in the solvent output from the device, and more preferably, thesorbent binds at least 70%, or more preferably 85%, or more preferably90%, or even more preferably 95%, and most preferably 99% of the matrixinterfering agents present in the sample.

In certain embodiments, the devices can be used in a combinationfiltration solid phase extraction mode (SPE). For example, the methodscan further conprise optionally conditioning the sorbent by washing thesorbent with at least one conditioning solvent or mixture of solventsprior to contacting the bioanalytical sample with the sorbent. Themethods can further comprise optionally washing the sorbent withadsorbed analytes and matrix interfering agents with a wash solvent ormixture of solvents to remove unbound components. In accordance withfurther SPE uses, the methods can further comprise eluting analytes fromthe sorbent with eluting solvents of sequentially increasing solventstrength to remove more nonpolar analytes without contaminating theanalytes with the adsorbed matrix interfering agents.

In an additional embodiment, a method is provided for reducing matrixeffects in a bioanalytical sample comprising at least 50% (v/v) proteindenaturing organic solvent, the method comprising: a) providing asorbent capable of binding matrix interfering agents present in thebioanalytical sample; b) contacting the bioanalytical sample with thesorbent for at least 10 seconds; and c) separating the solution from thesorbent, wherein the amount of matrix interfering agents in theresulting treated sample is reduced. Preferably, said contacting isperformed for from about 10 seconds to about 10 minutes, and the sorbentbinds at least 50% of the matrix interfering agents present in thebioanalytical sample, while providing recovery of at least 90% of theanalytes in the solvent output from the device. The method can furthercomprise contacting the bioanalytical sample with a filtering means forremoving precipitated protein particles. Preferably, the contacting witha filtering means and with the sorbent is done in the same step.

In additional embodiments, there is provided a method for preparing adevice for reducing matrix effects in a bioanalytical sample, comprisingthe following steps: a) providing a support capable of containing aquantity of sorbent and a filtering means; and b) providing an amount ofsorbent effective to retain matrix interfering agents and a filteringmeans effective to remove precipitated proteins present in the sample;and c) assembling the filtering means and the sorbent within thesupport.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and in part willbecome apparent to those skilled in the art upon examination of thefollowing, or may be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates several embodiments of the invention.

FIG. 2 shows LC-MS/MS traces showing the gradual buildup ofphospholipids (phosphatidylcholine) on a HPLC column over the course of50 injections of dilute plasma.

FIG. 3 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 4 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 5 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 6 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 7 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 8 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 9 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 10 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 11 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 12 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 13 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 14 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 15 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 16 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 17 shows chromatograms of samples subjected to treatment withsorbent relative to untreated samples.

FIG. 18 shows chromatograms of analytes and matrix interfering agents ona reversed phase sorbent.

FIG. 19 shows chromatograms of analytes and matrix interfering agents ona polar modified reversed phase sorbent.

FIG. 20 shows chromatograms of analytes and matrix interfering agents ona polar modified reversed phase sorbent.

FIG. 21 shows chromatograms of analytes and matrix interfering agents ona polar modified reversed phase sorbent.

FIG. 22 shows a bar chart illustrating turbidity of protein precipitatedsamples subjected to filtration through filters with varying pore sizes.

FIG. 23 shows the time dependence for removal of phosphatidylcholinefrom protein precipitated plasma samples by two sorbents.

FIG. 24 shows the time dependence for removal of Tween 80 from proteinprecipitated plasma samples by two sorbents.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions and Overview

Before the present invention is described in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto specific analytes, chromatographic methods, filtration andpurification structures, or the like, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to limit thescope of the present invention.

It must be noted that as usedherein and in the claims, the singularforms “a,” “and” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “an analyte”includes two or more analytes; reference to “a phospholipid” includestwo or more phospholipids, and so forth.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

As used herein the term “selectivity” refers to the ratio betweencorrected retention times (T_(r)) for analytes eluting at differentretention times. Corrected T_(r) is calculated by the following formula:T_(r)−T₀. where T₀ is the transit time for unretained species. Thesorbents utilized herein provide selectivity between matrix interferingagents and analytes of interest, i.e., the ratio of the corrected T_(r)for matrix interfering agents relative to the corrected T_(r) foranalytes is greater than 1.

As used herein, the term “analyte” or “analyte of interest” means anymolecule to be characterized, identified or quantitated in a sample ofbiological, organic, synthetic, natural or inorganic origin. Forexample, a candidate therapeutic compound or metabolite thereof can bean analyte, and can be present in, for example, a blood plasma sample,saliva, urine, drinking water, mixture of synthetic or natural products,or environmental sample. An analyte can exhibit any polarity, fromnonpolar to polar.

As used herein, the term “macropores” generally refers to pores withdiameters greater than about 0.05 μm; these are considered to be“throughpores” in the sense of allowing fluid flow through a monolithicfilter or sorbent. The term “mesopores” refers to pores with diametersbetween about 2 nm and 50 nm; and the term “micropores” refers to poreswith diameters less than about 2.0 nm.

The term “reversed phase” refers to a non-polar stationary phasecomprising alkyl or aromatic moeities providing a hydrophobic surfacefor adsorption of nonpolar compounds. A common reversed phase stationaryphase is a silica which has been treated with RMe₂SiCl, where R is astraight chain alkyl group such as C₁₈H₃₇ or C₈H₁₇. another reversedphase stationary phase is provided by polystyrene-divinylbenzene.

The term “polar modified reversed phase” refers to a non-polarstationary phase comprising alkyl or aromatic moeities providing ahydrophobic surface for adsorption of nonpolar compounds, wherein thestationary phase has been further modified to contain (or furthercomprises) a polar moiety such as an amide, ether, amino, carboxy,sulfonamide, and the like. Nonlimiting examples of polar modifiedreversed phase stationary phases are described in U.S. Pat. No.7,125,488 to Li, U.S. Pat. No. 7,056,858 to Kallury, and U.S. PatentApplication Publication Nos. 20060247361 and 20060247362 to Shah.

As used herein, the term “strongly polar” means a molecule that, basedon the octanol-water partition coefficient log P, has a log P value of−1.0 to +0.5.

As used herein, the term “moderately polar” means a molecule that, basedon the octanol-water partition coefficient log P, has a log P value of0.5 to 1.5.

As used herein, the term “nonpolar” means a molecule that based on theoctanol-water partition coefficient log P, has a log P value greaterthan or equal to 2.0.

Sorbent polar functionalities as used herein include but are not limitedto the following: —NRC(O)— (amide), —C(O)NR— (carbamyl), —OC(O)NR—(carbamate), —OC(O)R (alkyloxy), —NRC(O)O— (urethane), —NRC(O)NR—(carbamide or urea), —NCO (isocyanate), —CHOHCHOH— (diol), CH₂OCHCH₂O—(glycidoxy), —(CH₂CH₂O)_(s)— (ethoxy), —(CH₂CH₂CH₂O)_(s)— (propoxy),—C(O)— (carbonyl), —C(O)O— (carboxy), —CH₂C(O)CH₂— (acetonyl), —S—(thio), —SS— (dithio), —CHOH—, —O— (ether), —SO— (sulfinyl), —SO₂—(sulfonyl), —SO₃— (sulfonic acid), —OSO₃ (sulfate), —SO₂NR—(sulfonamide), —NR_(q)—, (amines), and —NR_(q) ⁺—, where R is not H(quaternary amines), —CN (nitrile), —NC (isonitrile), —CHOCH— (epoxy),—NHC(NH)NH— (guanidino), —NO₂ (nitro), —NO (nitroso), —OPO₃—(phosphate), —OH (hydroxy), and s is 1-12.

As used herein, the term “matrix effects” refers to any substancepresent in a sample that interferes with quantitation of an analyte.Matrix effects can be manifested in traditional chromatographicapplications by the co-elution of contaminating matrix constituents withanalytes of interest, causing interference with spectrophotometricquantitation methods, for example. Matrix effects are commonly observedin mass spectromertry applications, when ion suppression of an analyteis observed.

As used herein, the term “matrix interfering agent” refers to anysubstance present in a bioanalytical sample in relatively highconcentration (usually at least 1 mg/ml) that causes matrix effects,that is, interferes with quantitation of an analyte. Matrix interferingagents commonly suppress the ionization of a particular analyte presentin the sample during electrospray ionization for mass spectrometricanalysis. The relative abundance of the analyte can be underrepresentedand/or underestimated or overrepresented from its true abundance in thesample due to matrix effects.

The present invention is related to novel sample preparation devices andmethods of manufacturing and using them, such as devices for samplepreparation for LC/MS, solid phase extraction devices, and the like. Thepresent inventors have surprisingly discovered that a single device canprovide the dual functionality of removing precipitated proteins andmatrix interferences in one step, resulting in superior analyticalcapabilities and speeds and performance. Although practitioners havebeen extensively employing similar devices and methods for samplepreparation and the like, the present inventors are the first todiscover the combination of filter and sorbent functionality thatremoves precipitated protein (and other) particulates and that adsorbsmatrix interfering agents while selectively eluting analytes ofinterest, and to utilize these functionalities to produce productshaving superior performance, time saving, ease of use and manufacture,to the inventors' knowledge to date. The combination of filtering means,sorbents, and solvents allows a one step cleanup procedure for removingprecipitated proteins and matrix interfering agents, particularly plasmalipids, which results in not only increased convenience, but cleanersamples for analysis than had heretofore been possible, an unexpectedand surprising result.

Accordingly, there are provided devices for reducing matrix effects in aprotein precipitated bioanalytical sample comprising: a support, and asorbent associated with the support capable of binding matrixinterfering agents present in the bioanalytical sample, wherein thedevice further comprises filtering means for removing precipitatedprotein particles. The invention further provides methods for reducingmatrix effects and removing protein precipitates in a bioanalyticalsample, said methods comprising: a) providing a device comprising asupport, and a sorbent associated with the support, wherein said sorbentis characterized by a selectivity greater than 1 for matrix interferingagents relative to analytes of interest present in the bioanalyticalsample, and further comprising filtering means for removing proteinprecipitates present in the sample; b) contacting the bioanalyticalsample with the sorbent; and c) eluting the analytes from the sorbentwhile retaining the matrix interfering agents and precipitated proteins,wherein the amount of matrix interfering agents and proteins in theresulting treated sample is reduced.

Various aspects and embodiments of the invention will be described ingreater detail below.

II. Devices

The devices of the invention comprise a sorbent suitable for the removalof matrix interfering agents (e.g., surfactants, phospholipids,excipients, dosing agents, etc.) from protein precipitated samples incombination with a particulate filter means suitable for the removal ofprecipitated proteins or other unwanted solid matter. In certainembodiments, the particulate filter functionality and the sorbent arecombined into one component. In additional embodiments, the particulatefilter is a separate component from the sorbent component. In certainadditional embodiments, a particulate filter is not included whereprotein precipitates or other matter does not interfere with instrumentperformance or have been removed by other means.

Several different embodiments of the device are shown in FIG. 1. Typicaldevices include cartridges (e.g., inline cartridges, individual filtercartridges), pipette tips (with or without a protein precipitationfilter), multiple well plates of many varieties, syringe filters andinline columns, and the like. An exemplary embodiment is described inExamples 10 and 11, and demonstrates the dramatic improvement in samplepreparation procedures possible using the devices of the presentinvention.

The devices can include particulate porous or nonporous sorbents,monolithic sorbents, sorbent impregnated webs, fibers or filters, orother porous media with the proper selectivity. The sorbents can bepolymeric or silica based, as discussed further below. The devicesgenerally include a filtering means, such as size exclusion filters orsorbents providing a dual sorbent and filtering function. Thearrangement of filtering means and sorbent is generally unimportant, andcan be arranged as desired to suit a particular application. However,where no particles are present or have been removed by other means, thedevices can omit the filtering means.

A. Supports

As used herein, the terms “support” mean a porous or non-porous waterinsoluble material. A support or a supporting format can have any one ofa number of configurations or shapes, such as strip, plate, disk, rod,cylinder, well, cone, and the like. A support or supporting format canbe hydrophobic, hydrophilic or capable of being rendered hydrophilic,and can comprise inorganic powders such as silica, zirconia, andalumina; natural polymeric materials, synthetic or modified naturallyoccurring polymers, such as nitrocellulose, cellulose acetate,poly(vinyl chloride), polyacrylamide, polyacrylate, polyethylene,polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate,poly(ethylene terephthalate), nylon, poly(vinyl butyrate),polytetrafluoroethylene (PTFE or Teflon®), etc.; either used bythemselves or in combination, and in conjunction with other materialssuch as glass, ceramics, metals, and the like. The support can beconstructed of any material suitable for holding and dispensing liquids,and will generally be a polymeric material such as a polyolefin,fluorinated polymers, polysulfone, polyethersulfone, cellulose acetate,polystyrene, polystyrene/acrylonitrile copolymer, PVDF, and the like.Polyolefinic materials are preferred, for example, polypropylene,polyethylene, poly(tetrafluoroethylene), or copolymers thereof. Fornonaqueous liquids, the tube can be constructed of a material that willnot dissolve or leach contaminants into the nonaqueous liquid.Preferably, the devices are constructed from ultra-clean polymers,preferably polypropylene.

Support structures can be of any size or shape to suit the need of anyspecific application. For example, for sample pretreatment applications,the support can be in the form of individual filter cartridges ormultiwell plates, luer based syringe filters, pipette tips, as shown inFIG. 1, and the like. In one embodiment, the support can be a multi-wellfiltration or solid phase extraction apparatus such as described in U.S.Pat. No. 6,491,873 to Roberts. For chromatographic in line use, thesupport can be in the form of in-line columns for multiple or singleuse, and the like.

In certain embodiments, the support provides sufficient volume to allowprecipitation of proteins directly in the device (e.g., the deviceincludes a reservoir), obviating the need to perform the precipitationin one container and subsequently transfer the sample to the device forremoving matrix interfering agents and precipitated proteins. Forexample, to perform a protein precipitation using a 3:1 or 4:1 dilutionof the aqueous bioanalytical sample with a denaturing organic solvent,the device should include a reservoir having up to five times the volumeof the original sample volume.

For solid phase extraction applications, the support can be in the formof a syringe cartridge, pipette tip or the like. Theoretically, there isno limitation in size and shape, and the dimensions of the devices canbe determined entirely from the constraints of practical applications.For some applications, such as preparative scale applications, muchlarger devices might be used. Smaller structures may also becontemplated for micro-fabricated devices. The present invention can beapplied to make parts in any relevant size range requiring onlysufficient material and processing equipment of sufficient size tohandle the parts to be processed.

B. Sorbents

The device for reducing matrix effects in a bioanalytical samplecomprises a sorbent for selectively retaining matrix interfering agents.Preferably, the sorbent comprises a reversed phase or a polar modifiedreversed phase. The sorbent is capable of binding analytes and agentscontributing to matrix effects present in the bioanalytical sample. Thesorbent exhibits selective binding for analytes and matrix interferingagents, meaning that under the solvent conditions utilized herein, thesorbent retains matrix interfering agents while not retaining analytes,even relatively nonpolar analytes such as posaconazole (log P=5.66).Preferably, the sorbent binds at least 50% of the matrix interferingagents present in the bioanalytical sample while providing recovery ofat least 75% of the analytes in the solvent output from the device, andpreferably at least 90% of the analytes are recovered from the device.In more preferred embodiments, the sorbent binds at least 70%, or morepreferably 85%, or more preferably 90%, or even more preferably 95%, ormost preferably 99% of the matrix interfering agents.

Typical agents causing matrix effects in test samples includesurfactants and lipids, although any agent that causes matrix effects isencompassed in the present invention provided it can be reduced orremoved using the devices and methods described herein.

The amount of sorbent needed for a typical plasma sample depends on thebinding capacity of the sorbent. A typical plasma sample has a volumebetween 50 μl and 200 μl and therefore 20-30 mg of Polaris® (10 μmparticle size) C18-A is an appropriate amount of sorbent for this samplesize. When the sorbent is a glass fiber monolith with embedded particlessuch as Spec® disks (Varian, Inc., Palo Alto, Calif.), 4 to 8 disksprovide an appropriate amount of sorbent. One skilled in the art canreadily determine the desired amount of sorbent for a particularapplication. For example, plasma from different species of animals orfrom animals fed different diets contains varying amounts ofphospholipids. For higher concentrations of phospholipids present in thesample, a relatively larger amount of sorbent would be required formaximal removal of matrix interfering phospholipids.

Preferably, the sorbent is characterized by sufficient selectivitybetween matrix interfering agents and analytes of interest in thesolvent system being utilized in order to provide separation between theinterfering agents and analytes. In one embodiment, the sorbent exhibitsa selectivity between phosphatidylcholine and an analyte of interest ofat least 1.0 Depending on the particular sorbent, solvent conditions,matrix interfering agent and analyte of interest, a selectivity of atleast 1.1, more preferably 1.2, more preferably 1.3, more preferably1.4, or even more preferably 1.5 is provided. As shown in Example 6, useof a polar (amide) modified reversed phase sorbent provides optimalretention of matrix interfering agents and reduction in matrix effects,while allowing maximal recovery of analytes which elute ahead of thematrix interfering agents. Accordingly, a preferred sorbent is an amidemodified reversed phase.

Use of pH modifiers provides further control over the selectivitybetween matrix interfering agents and analytes of interest. Nonpolarbasic analytes can be selectively eluted (e.g., are not retained by thesorbent) by acidifying the precipitation solvent solution. Protonationof the base provides a more polar molecule less likely to be retained bythe nonpolar stationary phase provided by the sorbent. Similarly,basification of solutions containing acidic analytes leads to lessretention, and hence greater separation and enhanced selectivity,between the matrix interfering agents and the acidic analytes ofinterest. Example 4 and Tables 10 and 11 further illustrate thisprinciple in the operation of the devices and methods of the presentinvention.

Any sorbent known in the art can in principle be utilized in the devicesand methods described herein. Sorbents can include polymeric sorbents,inorganic sorbents, hybrid organic-inorganic sorbents, bonded phases,and combinations thereof.

In one embodiment, the sorbent comprises a monolithic or particulateinorganic substrate that is modified to produce a bonded phase with atleast one silane having the formula

R¹ _(δ)-Q_(α)-(CH₂)_(β)SiR² _(γ)X_(3−γ),

wherein R¹ is hydrogen, C₁-C₁₀₀ substituted or unsubstitutedhydrocarbyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; whereinthe substituents are selected from C₁-C₁₂ hydrocarbyl, hydroxyl, alkoxy,halogen, amino, nitro, sulfo, and carbonyl; α is 0 or 1; β is 0-30; γ is0, 1 or 2; δ is 0-3; R² is C₁-C₁₀₀ substituted or unsubstitutedhydrocarbyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; whereinthe substituents are selected from C₁-C₁₂ hydrocarbyl, hydroxyl, alkoxy,halogen, amino, nitro, sulfo, and carbonyl; Q is independently selectedfrom —NHC(O)—, —C(O)NH—, —OC(O)NH—, —NHC(O)O—, —NHC(O)NH—, —NCO,—CHOHCHOH—, CH₂OCHCH₂O—, —(CH₂CH₂O)_(n)—, —(CH₂CH₂CH₂O)_(n)—, —C(O)—,—C(O)O—, —OC(O)—, CH₃C(O)CH₂—, —S—, —SS—, —CHOH—, —O—, —SO—, —SO₂—,—SO₃—, —OSO₃—, —SO₂NH—, —SO₂NMe-, —NH—, —NMe-, —NMe₂ ⁺—,—N[(CH₂)_(n)]₂—, —CN, —NC, —CHOCH—, —NHC(NH)NH—, —NO₂, —NO, —OPO₃—,where n is 1-30; and X is a leaving group, as described in U.S. Pat. No.7,125,488 to Li.

Preferably, the inorganic substrate comprises a metal-oxide or metalloidoxide, such as silica, alumina, zeolite, mullite, zirconia, vanadia ortitania, or mixtures or composites thereof, having reactive metal oxidescapable of reacting with an alkoxysilane, aminosilane, hydroxysilane orhalosilane. The inorganic substrate can take the form of beads orregular or irregular particles ranging in size from about 0.001 mm to 10mm in diameter, preferably 0.005 to 0.04 mm, fibers (hollow orotherwise) of any size, membranes, flat surfaces ranging in thickness,for example, from about 0.1 mm to about 10 mm thick, and sponge-likematerials, such as frits or monoliths with pores from 0.05 microns toseveral mm in diameter. After modification of the inorganic substratesurface with a silane, the silane is covalently attached to theinorganic substrate via an oxygen linkage to produce a bonded phase.

In certain embodiments, the modified inorganic substrate is a bondedalkyl phase such as C8 or C18 (where α is 0), useful in reversed phaseadsorption and chromatographic applications. In certain preferredembodiments, α is 1, and the modified inorganic substrate is a bondedpolar embedded reversed phase, suitable for enhanced retention of matrixinterfering agents without retaining nonpolar analytes under solventconditions of at least 50% (v/v) organic solvent. In a particularlypreferred embodiment, the modified inorganic substrate comprises a C18bonded phase having a polar embedded amido functionality, such asPolaris® C-18 A (available through Varian, Inc., Palo Alto, Calif.). Inanother particularly preferred embodiment, the modified inorganicsubstrate comprises a C18 bonded phase in which remaining silanol groupsare further reacted with an amido functionalized endcapping reagent,such as Polaris® C-18 Amide (available through Varian, Inc., Palo Alto,Calif.). In additional embodiments, the modified inorganic substratecomprises a C8 bonded phase, or an ether functionality, such as Polaris®C18-Ether or Polaris® C8-Ether (Varian, Inc., Palo Alto, Calif.). One ofordinary skill will recognize that any of the bonded phases of varyingcompositions described in U.S. Pat. No. 7,125,488 to Li can be utilizedin the inventive devices and methods described herein.

In an additional embodiment, the device comprises a polymeric sorbentdescribed in U.S. Pat. Nos. 7,056,858 and 6,926,823 to Kallury. Thissorbent comprises: (i) a polymeric backbone adapted to form at least oneof a dipolar interaction and a hydrophobic interaction; and (ii) anamide functionality associated with the backbone and adapted to undergoproton accepting and proton donating interactions. Preferably the amidefunctionality is associated with the backbone via a covalent bond. Apolymeric sorbent of the present invention also comprises an amidefunctionality associated with the polymeric backbone and adapted toundergo one or more interactions selected from the group consisting ofproton accepting, proton donating and dipolar interactions, for example,with the functionalities of an analyte. Representative amidefunctionalities include acetamide, N-alkylamides, N-aryl-amides andN-heteroaryl amides.

Any polymer adapted to form at least one of a dipolar interaction and ahydrophobic interaction can be employed as a polymeric backbone in thisembodiment. A polymeric backbone can comprise, for example, poly(styrenedivinylbenzene), copolymers of styrene or divinylbenzene withfunctionalized styrenes or heterocycles carrying substituents such ashalo, alkoxy, ester or nitro; or copolymers such as (but not restrictedto) polystyrene-polyacrylamide and polystyrene-polyacrylates. Thus, arepresentative, but non-limiting, list of polymers that can be employedas a polymeric backbone in a sorbent includes, but is not limited to,poly(styrene divinylbenzene), copolymers comprising styrene ordivinylbenzene and methylmethacrylate, halogenated or nitrated oraminated or hydroxylated styrenes, functionalized isocyanurates,urethanes, acrylamides or acrylonitriles and functionalized heterocyclicsystems, such as vinyl/allyl pyridines. In one embodiment, a polymericbackbone comprises poly(styrene divinylbenzene). In certain embodiments,it is preferable that the polymeric backbone comprises spherical ornon-spherical particles having a diameter of between about 0.001 mm andabout 10 mm in diameter, preferably from about 0.005 to about 0.04 mm indiameter.

In other embodiments, a polymeric sorbent can be utilized in a polymermodified porous substrate format, for example, formed on a poroussubstrate in the form of a monolith, agglomerated particles, or woven ornonwoven fibers, (e.g., glass or polymer fibers). Preferably, thepolymer modified porous substrate comprises a porous substrate and apolymeric monolith formed thereon, wherein the polymeric monolith hasthe formula

wherein A is selected from C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl, optionally substituted with -L-Q_(p)-R_(q); q is 0-3; p is0-5; Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—, —OC(O)R, —NRC(O)O—, —NRC(O)NR—,—NCO, —CHOHCHOH—, CH₂OCHCH₂O—, —(CH₂CH₂O)_(s)— and —(CH₂CH₂CH₂O)_(s)—,—C(O)—, —C(O)O—, —CH₂C(O)CH₂—, —S—, —SS—,—CHOH‘3, —O', —SO—, —SO₂—,SO₃—, —OSO₃, —SO₂NR—, —NR_(q)—, and —NR_(q) ⁺—, where R is not H, —CN,—NC, —CHOCH—, —NHC(NH)NH—, —NO₂ , —NO, 'OPO₃—, —OH, where s is 1-12; and

-   R is hydrogen, C₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl,    C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl;

P is

L is a bond or a C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl; andwherein the order of [—CH₂—CR-L-A-P] and [—CH₂—CR-L-A] is random, blockor a combination thereof, described in U.S. Patent ApplicationPublication No. 20060247352 to Shah.

In yet other embodiments, a polymeric sorbent can be utilized in apolymer modified porous substrate format, for example, formed on aporous substrate in the form of a monolith, agglomerated particles, orwoven or nonwoven fibers, (e.g., glass or polymer fibers). Preferably,the polymeric sorbent is a polar functionalized polymer modified poroussubstrate, comprising a porous substrate and a polar functionalizedpolymeric monolith formed thereon, wherein the polymeric monolith hasthe formula

wherein A is selected from C₅₋₁₀ monocyclic or bicyclic aryl orheteroaryl, optionally substituted with C₁₋₁₂ branched or unbranchedhydrocarbyl, or halo; wherein n/m is from about 0.001 to about 1000;wherein r is 0 or 1; wherein Q is —NRC(O)—, —C(O)NR—, —OC(O)NR—,—OC(O)R, —NRC(O)O—, —NRC(O)NR—, —NCO, —CHOHCHOH—, CH₂OCHCH₂O—,—(CH₂CH₂O)_(n)— and —(CH₂CH₂CH₂O)_(s)—, where s is 1-12, —C(O)—,—C(O)O—, —CH₂C(O)CH₂—, —S—, —SS—, —CHOH—, —O—, —SO—, —SO₂—, —SO₃—,—OSO₃, —SO₂NR—, —NR_(q)—, and —NR_(q) ⁺—, —CN, —NC, —CHOCH—,—NHC(NH)NH—, —NO₂, —NO, —OPO₃—, —OH, or combinations thereof; L is abond, or a C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl;

-   R is hydrogen, C₅₋₁₀ monocyclic or bicyclic aryl or heteroaryl,    C₁₋₁₂ branched, unbranched, or cyclic hydrocarbyl, optionally    substituted with halo, nitro, or alkyl;

P is

and wherein the order of [—CH₂—CR-L-A-P_(r)] and [—CH₂—CR-L-Q-R—P_(r)]is random, block or a combination thereof, described in U.S. PatentApplication Publication No. 20060247361 to Shah.

In addition, the polymeric sorbents described above can be utilized inthe present devices and methods without a porous substrate, for example,formed as beads or fibers. In yet other embodiments, the sorbent can bea polymeric sorbent as described in U.S. Pat. Nos. 6,576,767 and6,825,269 to Gottschall and available through instrAction GmbH(Ludwigshafen, DE), which describe the preparation of polymeric networkshaving functional groups on silica. The performance of certain of thesesorbents is illustrated in Examples 2 and 4.

In yet other embodiments, the sorbent can be a functionalized monolithicsorbent, comprising a glass fiber matrix embedded with a bonded phasecomprising metal oxide or metalloid oxide particles having reactivemetal oxides capable of reacting with silanes, such as alkoxysilanes,aminosi lanes, hydroxysilanes or halosilanes, such as described in U.S.Patent Application Publication No 20060216206 to Hudson. Suitable metaloxides and metalloid oxides include silica, alumina, zeolite, mullite,zirconia, vanadia or titania, or mixtures or composites thereof,preferably silica. Likewise, the glass fiber matrix is composed of ametal or metalloid oxide. After reaction with a silane, the silane iscovalently attached to silica particle via an oxygen linkage, and themetal or metalloid oxides are functionalized by, for example,hydrocarbyl, amido, carbamyl, carbamato, urethane, carbamido,isocyanato, diol, glycidoxy, ethoxy, propoxy, carbonyl, carboxy,acetonyl, thio, dithio, hydroxy, ether, sulfinyl, sulfonyl, sulfonicacid, sulfate, sulfonamido, amino, nitrilo, isonitrilo, epoxy,guanidino, nitro, nitroso, and phosphate.

The silica can be chemically treated (or functionalized) by any methodknown in the art. In one embodiment, the silica is bonded with alkylmoieties, typically C₂₋₃₀ alkyl groups, to render the silica hydrophobicand provides a reversed phase for adsorption of hydrophobic compounds.In another embodiment, the silica is bonded with a C18 bonded phase withan amide endcapping reagent or a C18 bonded phase having a polarembedded moiety and provides a polar modified reverse phase. Any bondedphase that can be used to modify silica is possible, such as amino,cyano, glycidyl, and the like, as well as anion or cation exchangegroups, as discussed above. In a preferred embodiment, thefunctionalized monolithic sorbent is comprised of glass microfibersimpregnated with modified silica, preferably prepared using organosilanechemistries (available from Varian, Inc., Palo Alto, Calif.), which aresimilar to the SPEC® product. This monolithic bonded silica allowsgreatly improved flow and much less void volume and less solvent is usedin sample processing. The binding capacity of the functionalizedmonolithic sorbent is generally in the range of about 1 microgramanalyte per 0.1 mg sorbent. The glass fiber matrix material typically isconstructed from randomly distributed fibers which create a tortuouspath of nominally rated size, and has a thickness of from about 0.1 mmto about 2 mm, more typically about 1 mm. In a preferred embodiment, thesorbent is that utilized in Spec® IQe (Varian, Inc., Palo Alto, Calif.).

In additional embodiments, the sorbent can comprise a phospholipotropicmultivalent cation coupled to a support, such as described in U.S.Patent Application Publication No. 20050054077 (Bennett, et al.) forremoving phospholipids from biological samples. Such sorbents comprisetransition metals, lanthanides or actinides, preferably cerium, foradsorbing phosphate containing compounds such as phospholipids.

C. Filtering Means

Particles sizes vary in protein precipitated samples depending onprotein concentration and composition, protein precipitation methodused, and the like. The use of various denaturing solvents with plasmasamples is typical in the analytical laboratory, and particles to beremoved from plasma samples likewise vary. In another aspect, thedevices and methods described herein provide a filtering means forremoving protein precipitated particles from a bioanalytical sample. Inespecially preferred embodiments, the devices and methods remove proteinparticulates resulting from precipitation procedures such as theaddition of denaturing solvents or salts.

The particle sizes of the protein precipitate can vary depending oncomposition, method of precipitation and other variables, and thus thefiltering means can be chosen to provide the desired particle sizeremoval. For example, when using ACN, precipitated protein particlediameters are generally larger, and therefore, filters having pore sizesof about 0.2 μm to about 0.45 μm are generally sufficient to removeunwanted particulate contaminants; when using MeOH, particle diameterstend to be smaller, and therefore pore sizes in filters of 0.1 μm to 0.2μm or less are typically sufficient. As shown in Example 7, plasmasamples precipitated with 75% (v/v) MeOH/3% formic acid produced smallerparticle sizes which could be removed using 0.2 μm pore size Captiva®filters. In contrast, plasma samples precipitated with 75% (v/v) ACNproduced larger particle sizes that could be removed using 0.45 μm poresize Captiva® filters.

In certain embodiments, the filtering means is effective to provideoptical clarity to the protein precipitated bioanalytical sample (e.g.,(% T at 524 nm>95). Preferably, the filtering means is characterized inhaving pore sizes between about 0.05 μm and about 0.5 μm in diameter forremoving precipitated protein particles present in the sample. Incertain preferred embodiments, the filtering means is characterized inhaving pore sizes between about 0.1 μm and about 0.2 μm in diameter. Inparticularly preferred embodiments, the filtering means is a sizeexclusion membrane excluding particles greater than 0.2 μm and 0.45 μmin diameter from passage through the filter.

In alternative embodiments, the filtering means is an inorganic monolithhaving a maximum pore size less than or equal to the diameter of theparticles to be removed from the sample, e.g., having pore sizes betweenabout 0.05 μm and about 0.5μm in diameter or more preferably betweenabout 0.1 μm and about 0.2 μm in diameter. In certain other preferredembodiments, the filtering means is integral with the sorbent orassociated with the sorbent, for example, a porous inorganic monolithhaving macropores of a diameter sufficiently small so as to excludeparticles from the sample, and comprising a reversed phase or polarmodified reversed phase bonded to the porous inorganic monolith.

Preferably, the filtering means is a size exclusion filter such as aCaptiva® filter or a Captiva® plate (96 well format). The size exclusionfilter can comprise any material known in the art that is stable to thesolvent conditions utilized in the protein precipitation and removal ofmatrix interfering agents. Suitable materials for a size exclusionfilter include, without limitation, polyolefinic materials such aspolypropylene, polyethylene, poly(4-methylbutene), or copolymersthereof, nylon, PTFE, fluorinated polymers, polysulfone,polyethersulfone, cellulose acetate, polystyrene,polystyrene/acrylonitrile copolymer, PVDF, nitrocellulose, poly(vinylchloride), polyacrylamide, polyacrylate, polystyrene, polymethacrylate,poly(ethylene terephthalate), poly(vinyl butyrate), etc.; either used bythemselves or in combination, and in conjunction with other materialssuch as glass, ceramics, metals, and the like. The size exclusionfilters should be constructed from materials that will not dissolve orleach contaminants into the sample. Preferably, the filters areconstructed from ultra-clean polymers, preferably polypropylene, PVDF,or PTFE. In a preferred embodiment, the filtering means is a PTFEfilter, which provides the added benefit of retaining the solvent untilthe user initiates elution of the sample through the filter (e.g., byapplying pressure, a vacuum or centrifugal force), providing improvedcontrol over timing and providing additional ease of use.

In additional embodiments, the filtering means can be a polymeric orinorganic monolith (as well as a size exclusion filter) having a maximumpore size sufficient to remove the desired size particles. Typically,the maximum pore size that is sufficient to remove the desired sizeparticles is smaller than the diameter of the particles to be removedfrom the sample. However, in monolithic filter means providing atortuous path for fluid flow through throughpores (macropores), themaximum pore size that is sufficient to remove the desired sizeparticles may be larger than the diameter of the particles to be removedfrom the sample. In preferred embodiments, the filtering means is aporous glass monolith, for example, prepared as described in U.S. PatentApplication Publication No. 20060131238 to Xu. One skilled in the artcan readily determine the size of pores desirable to remove particles ofa given size from samples, for example, resulting from proteinprecipitation in different solvents, salts or acids. One skilled in theart can also readily prepare monolithic filtering means for removingparticles of a chosen size.

D. Protein Precipitation Treatments

The skilled artisan will be aware of numerous protein precipitationtreatments that can be utilized in the methods of the present invention.Typical treatments that can be utilized to precipitate proteins includeacid treatment (e.g., trichloroacetic acid, formic acid, etc.),denaturing solvents (e.g., methanol, acetonitrile, acetone, etc.), heattreatment (resulting in denaturation of the proteins), salt treatments(e.g., ammonium sulfate), and combinations thereof. The devices andmethods described herein are preferably utilized with samples whereinthe protein precipitation is effected by the addition of denaturingsolvents (usually at least 2:1 organic solvent to water). For example, a2:1 or a 3:1 dilution of an aqueous sample with an organic solvent suchas methanol, acetone, or acetonitrile, will result in the precipitationof proteinaceous components found in bioanalytical samples such asplasma, cell culture supernatants, tissue extracts, tissue homogenates,and the like. Thus, precipitated samples typically comprise 50% (v/v) orgreater organic phase (e.g., 66% (v/v) for 2:1 dilution with solvent or75% (v/v) for 3:1 dilution with solvent). In preferred embodiments, theproteins are precipitated using a combination of acid treatment andsolvent dilution (e.g., 2:1 methanol, 1% formic acid). The inclusion ofacid is advantageous when the analytes of interest are basic analytes,as is typical when performing pharmacokinetic analyses and the like. Oneskilled in the art will recognize that precipitation conditions will betailored to the particular analytes of interest and nature of theparticular bioanalytical samples. Under certain conditions, it can beadvantageous to perform the precipitation in a basic solution, forexample, by including a base such as ammonium formate, etc. in theprecipitation solution.

E. Matrix Interfering Agents

Matrix interfering agents include a wide variety of substances that canbe present in a bioanalytical sample in the relatively highconcentration (usually at least 1 mg/ml) necessary to suppress theionization of an analyte present in the sample during electrosprayionization for mass spectrometric analysis. Matrix interfering agentsexert suppression of analyte detection that is very analyte dependent,meaning that detection of one analyte may be affected more adverselythan detection of another.

Commonly encountered matrix interfering agents include surfactants andother agents added to samples, lipids present in serum, excipients(dosing agents) added to a drug formulation, and the like. For example,NP 40 can be added to cultured cells to lyse the cells prior to analysisof a sample. Blood plasma from patients or experimental animals is knownto contain significant amounts of lipids such as cholesterol,triglycerides, phospholipids, lysophospholipids, lipoproteins, and thelike, all of which can be present at sufficiently high concentrations toexert matrix interfering effects, and/or to contaminate analyticalinstrumentation. Polyethylene glycol (PEG), surfactants, disintegrants,and other excipients can be present in dissolution studies of drugformulations.

Surfactants and plasma lipids are some of the most common matrixinterfering agents encountered in bioanalytical testing. Plasma lipidsthat can be removed using the presently described devices and methodsinclude cholesterol, cholesterol esters, triglycerides, phospholipids,lysophospholipids, lipoproteins, and the like, without limitation, solong as the sorbent exhibits a selectivity for the plasma lipid overanalytes of interest that is greater than 1. A preferred plasma lipid isphosphatidylcholine (PC), which can be present as the lyso form (havingonly one acyl chain), or the diacyl form (having two acyl chains).

Surfactants that can be removed using the presently described devicesand methods include a wide variety of surfactants, including nonionicsurfactants as well as ionic surfactants, including cationicsurfactants, anionic surfactants or zwitterionic surfactants. Nonionicsurfactants include, for example, polyoxyl stearates such as polyoxyl 40stearate, polyoxyl 50 stearate, polyoxyl 100 stearate, polyoxyl 12distearate, polyoxyl 32 distearate, and polyoxyl 150 distearate, andother Myrj™ series of surfactants, triblock co-polymers of ethyleneoxide/propylene oxide/ethylene oxide, also known as poloxamers, havingthe general formula HO(C₂H₄O)_(a)(—C₃H₆O)_(b)(C₂H₄O)_(a)H, availableunder the tradenames Pluronic and Poloxamer, sugar ester surfactants,sorbitan fatty acid esters such as sorbitan monolaurate, sorbitanmonopalmitate, sorbitan monostearate, sorbitan tristearate, and otherSpan™ series surfactants, glycerol fatty acid esters such as glycerolmonostearate, polyoxyethylene derivatives such as polyoxyethylene ethersof high molecular weight aliphatic alcohols (e.g., Brij 30, 35, 58, 78and 99) polyoxyethylene stearate (self emulsifying), polyoxyethylene 40sorbitol lanolin derivative, polyoxyethylene 75 sorbitol lanolinderivative, polyoxyethylene 6 sorbitol beeswax derivative,polyoxyethylene 20 sorbitol beeswax derivative, polyoxyethylene 20sorbitol lanolin derivative, polyoxyethylene 50 sorbitol lanolinderivative, polyoxyethylene 23 lauryl ether, polyoxyethylene 2 cetylether with butylated hydroxyanisole, polyoxyethylene 10 cetyl ether,polyoxyethylene 20 cetyl ether, polyoxyethylene 2 stearyl ether,polyoxyethylene 10 stearyl ether, polyoxyethylene 20 stearyl ether,polyoxyethylene 21 stearyl ether, polyoxyethylene 20 oleyl ether,polyoxyethylene 40 stearate, polyoxyethylene 50 stearate,polyoxyethylene 100 stearate, polyoxyethylene derivatives of fatty acidesters of sorbitan such as polyoxyethylene 4 sorbitan monostearate,polyoxyethylene 20 sorbitan tristearate, and other Tween™ series ofsurfactants, phospholipids and phospholipid fatty acid derivatives suchas fatty amine oxides, fatty acid alkanolamides, propylene glycolmonoesters and monoglycerides, such as hydrogenated palm oilmonoglyceride, hydrogenated soybean oil monoglyceride, hydrogenated palmstearine monoglyceride, hydrogenated vegetable monoglyceride,hydrogenated cottonseed oil monoglyceride, refined palm oilmonoglyceride, partially hydrogenated soybean oil monoglyceride, cottonseed oil monoglyceride sunflower oil monoglyceride, sunflower oilmonoglyceride, canola oil monoglyceride, succinylated monoglycerides,acetylated monoglyceride, acetylated hydrogenated vegetable oilmonoglyceride, acetylated hydrogenated coconut oil monoglyceride,acetylated hydrogenated soybean oil monoglyceride, glycerolmonostearate, monoglycerides with hydrogenated soybean oil,monoglycerides with hydrogenated palm oil, succinylated monoglyceridesand monoglycerides, monoglycerides and rapeseed oil, monoglycerides andcottonseed oils, monoglycerides with propylene glycol monoester sodiumstearoyl lactylate silicon dioxide, diglycerides, triglycerides,polyoxyethylene steroidal esters, Triton-X series of surfactantsproduced from octylphenol polymerized with ethylene oxide, where thenumber “100” in the trade name is indirectly related to the number ofethylene oxide units in the structure, (e.g., Triton X-100™ has anaverage of N=9.5 ethylene oxide units per molecule, with an averagemolecular weight of 625) and having lower and higher mole adductspresent in lesser amounts in commercial products, as well as compoundshaving a similar structure to Triton X-100™, including Igepal CA-630™and Nonidet P-40M (NP-40™, N-lauroylsarcosine, Sigma Chemical Co., St.Louis, Mo.), and the like. Any hydrocarbon chains in the surfactantmolecules can be saturated or unsaturated, hydrogenated orunhydrogenated.

Sugar ester surfactants include sugar fatty acid monoesters, sugar fattyacid diesters, triesters, tetraesters, or mixtures thereof, althoughmono- and di-esters are most preferred. Preferably, the sugar fatty acidmonoester comprises a fatty acid having from 6 to 24 carbon atoms, whichmay be linear or branched, or saturated or unsaturated C₆ to C₂₄ fattyacids. The C₆ to C₂₄ fatty acids are preferably chosen from stearates,behenates, cocoates, arachidonates, palmitates, myristates, laurates,carprates, oleates, laurates and their mixtures, and can include even orodd numbers of carbons in any subrange or combination. Preferably, thesugar fatty acid monoester comprises at least one saccharide unit, suchas sucrose, maltose, glucose, fructose, mannose, galactose, arabinose,xylose, lactose, sorbitol, trehalose or methylglucose. Disaccharideesters such as sucrose esters are most preferable, and include sucrosecocoate, sucrose monooctanoate, sucrose monodecanoate, sucrose mono- ordilaurate, sucrose monomyristate, sucrose mono- or dipalmitate, sucrosemono- and distearate, sucrose mono-, di- or trioleate, sucrose mono- ordilinoleate, sucrose polyesters, such as sucrose pentaoleate,hexaoleate, heptaoleate or octooleate, and mixed esters, such as sucrosepalmitate/stearate.

Sugar ester surfactants include those sold by the company Croda Inc ofParsippany, N.J. under the names Crodesta F10, F50, F160, and F110denoting various mono-, di- and mono/di ester mixtures comprisingsucrose stearates, manufactured using a method that controls the degreeof esterification, such as described in U.S. Pat. No. 3,480,616, thosesold by the company Mitsubishi under the name Ryoto Sugar esters, forexample under the reference B370 corresponding to sucrose behenateformed of 20% monoester and 80% di-, tri- and polyester, sucrose mono-and dipalmitate/stearate sold by the company Goldschmidt under the name“Tegosoft PSE”, sugar esterspresent in admixture with another compoundnot derived from sugar; such as the mixture of sorbitan stearate and ofsucrose cocoate sold under the name “Arlatone 2121” by the company ICI,other sugar esters such as, for example, glucose trioleate, galactosedi-, tri-, tetra- or pentaoleate, arabinose di-, tri- or tetralinoleateor xylose di-, tri- or tetralinoleate, or mixtures thereof. Other sugaresters of fatty acids include esters of methylglucose include thedistearate of methylglucose and of polyglycerol-3 sold by the companyGoldschmidt under the name of Tegocare 450. Glucose or maltosemonoesters can also be included, such as methylO-hexadecanoyl-6-D-glucoside and O-hexadecanoyl-6-D-maltose. Certainother sugar ester surfactants include oxyethylenated esters of fattyacid and of sugar include oxyethylenated derivatives such as PEG-20methylglucose sesquistearate, sold under the name “Glucamate SSE20”, bythe company Amerchol.

III. Methods for Bioanalytical Sample Preparation

The invention further provides methods for preparing a sample comprisingmatrix interfering agents and proteins for analysis. One skilled in theart will recognize that the devices and methods of the present inventioncan be implemented in various fashions to remove precipitated proteinsand reduce matrix effects. Typical analyses include chromatographic,spectrophotometric, mass spectrometric, and the like, and combinationsthereof. For example, an exemplary analysis method in the bioanalyticalarts for determining pharmaceutical analytes is LC/MS-MS.

Accordingly, there are provided methods for reducing matrix effects andremoving protein precipitates in a bioanalytical sample, said methodscomprising: a) providing a device comprising a support, and a sorbentassociated with the support, wherein said sorbent is characterized by aselectivity greater than 1 for matrix interfering agents relative toanalytes of interest present in the bioanalytical sample, and furthercomprising filtering means for removing protein precipitates present inthe sample; b) contacting the bioanalytical sample with the sorbent; andc) eluting the analytes from the sorbent while retaining the matrixinterfering agents and precipitated proteins, wherein the amount ofmatrix interfering agents and proteins in the resulting treated sampleis reduced. In certain embodiments, the method can further compriseprecipitating the proteins in the bioanalytical sample in the deviceprior to or simultaneously with the step of contacting the bioanalyticalsample with the sorbent. Preferably, step c) is performed using negativepressure (i.e., applying a vacuum), electrokinetic or centrifugal force,gravity- or capillary-driven or positive pressure to cause the sample topass through the sorbent and the filtering means, thereby removingmatrix interfering agents and precipitated proteins. For example,negative pressure can be used to pull samples through 96 well plates orcartridges using vacuum manifolds commerically available. Positivepressure can be used to push samples through, without limitation, an inline column or single use cartridge, pipette tips, or a luer basedsyringe filter. Similarly, centrifugal force can be used (a centrifuge)to push samples through filter cartridges or 96 well plates and thelike.

Preferably, the filtering means is characterized in having pore sizesbetween about 0.05 μm and about 0.5 μm in diameter for removingprecipitated protein particles present in the sample, and in certainembodiments, the filtering means comprises pore sizes between about 0.1μm and about 0.2 μm. In particular embodiments, the filtering meanscomprises pores sizes of 0.1, 0.2 and 0.45 μm. Preferably, the matrixinterfering agents are surfactants, lipids, excipients, or dosingagents, and in preferred embodiments, the lipids are phospholipids, andthe surfactants are selected from anionic surfactants or nonionicsurfactants. Preferably the surfactants comprise a hydrocarbon chainwhich can be advantageously retained using the sorbents describedherein. Preferably, the sorbent is characterized by sufficientselectivity between the matrix interfering agents and analytes ofinterest to provide retention of the matrix interfering agents whileproviding elution of the analytes of interest. In certain embodiments,the sorbent comprises a reversed phase or a polar modified reversedphase. In particular embodiments, there are provided methods forreducing matrix effects and removing precipitated proteins in a proteinprecipitated bioanalytical sample comprising matrix interfering agentsand analytes of interest, the method comprising passing the samplethrough the devices described herein.

In additional embodiments, methods are provided for reducing matrixeffects in a protein precipitated bioanalytical sample comprising matrixinterfering agents and analytes of interest, the method comprising: a)providing a device comprising a support, and a sorbent associated withthe support, wherein said sorbent is characterized by a selectivitygreater than 1 for matrix interfering agents relative to analytes ofinterest present in the bioanalytical sample; b) contacting thebioanalytical sample with the sorbent; and c) eluting the analytes fromthe sorbent while retaining the matrix interfering agents, wherein theamount of matrix interfering agents in the resulting treated sample isreduced. The device can further comprise filtering means for removingprecipitated protein particles. In preferred embodiments, when thebioanalytical sample comprises at least 50% (v/v) denaturing organicsolvent, the sorbent retains matrix interfering agents while notretaining analytes of interest. In additional embodiments, the sorbentretains matrix interfering agents while not retaining analytes ofinterest even at 66%, 75%, or even 90% (v/v) organic solvent, or in thepresence of pH modifiers (e.g., acids, bases). Preferably, the sorbentbinds at least 50% of the matrix interfering agents present in thebioanalytical sample, while providing recovery of at least 90% of theanalytes in the solvent output from the device, and more preferably, thesorbent binds at least 70%, or more preferably 85%, or more preferably90%, or even more preferably 95%, and most preferably 99% of the matrixinterfering agents present in the sample.

In certain embodiments, the devices can be used in a combinationfiltration and solid phase extraction mode (SPE). For example, themethods can further comprise optionally conditioning the sorbent bywashing the sorbent with at least one conditioning solvent or mixture ofsolvents prior to contacting the bioanalytical sample with the sorbent.The methods can further comprise optionally washing the sorbent withadsorbed analytes and matrix interfering agents with a wash solvent ormixture of solvents to remove unbound components. In accordance withfurther SPE uses, the methods can further comprise eluting analytes fromthe sorbent with eluting solvents of sequentially increasing solventstrength to remove more nonpolar analytes without contaminating theanalytes with the adsorbed matrix interfering agents.

In an additional embodiment, a method is provided for reducing matrixeffects in a bioanalytical sample comprising at least 50% (v/v) proteindenaturing organic solvent, the method comprising: a) providing asorbent capable of binding matrix interfering agents present in thebioanalytical sample; b) contacting the bioanalytical sample with thesorbent for at least 10 seconds; and c) separating the solution from thesorbent, wherein the amount of matrix interfering agents in theresulting treated sample is reduced. Preferably, said contacting isperformed for from about 10 seconds to about 10 minutes, and the sorbentbinds at least 50% of the matrix interfering agents present in thebioanalytical sample, while providing recovery of at least 90% of theanalytes in the solvent output from the device. The method can furthercomprise contacting the bioanalytical sample with a filtering means forremoving precipitated protein particles. Preferably, the contacting witha filtering means and with the sorbent is done in the same step.

In another embodiment, a sample is subjected to a protein precipitationtreatment followed by centrifugation, or does not contain sufficientprotein to warrant removal prior to analysis, and subsequently istreated with the sorbent with selectivity for matrix interfering agents,e.g., the supernatant is transferred from the protein pellet using apipette tip loaded with the sorbent. A pipette tip implementationsuitable for performing the method is depicted in FIG. 1.

In preferred embodiments, the method is utilized to reduce matrixeffects in a sample containing analytes of varying log P, butparticularly log P values over 2, the sorbent is a polar modifiedreversed phase sorbent and is used in high organic solvent strengths(e.g., 50% (v/v) to 95% (v/v)) with or without a pH modifier and with afilter pore size of 0.2 μm. For example, the method can compriseperforming a precipitation using 2:1 or 3:1 volume dilutions ACN or 3:1MeOH for plasma samples containing analytes having log P values≦5.2 andapproximately 20 mg-30 mg sorbent such as Polaris® C18-A or C18 Amide toremove particulates of protein and phosphatidylcholines and surfactants.With the higher amount of sorbent, most of the lysophosphatidylcholinescan be removed.

In additional preferred embodiments, the method is utilized to reducematrix effects in a sample containing analytes of varying log P, thesorbent (20-30 mg) is a reversed phase sorbent or a polar modifiedreversed phase sorbent and is used with 66% (v/v) organic solventstrengths and a filter pore size of 0.1-0.2 μm. For example, the methodcomprises performing a precipitation using 2:1 MeOH with 3% formic acidfor plasma samples containing analytes to remove particulates of proteinand substantially all phosphatidylcholines, includinglysophosphatidylcholines, and surfactants such as Tween 80 and SDS. Thisamount of sorbent (20 mg) is sufficient to remove 5 mg/ml surfactantswithout affecting recovery of analytes from plasma samples(approximately less than 1 ml). Aspects of this embodiment areillustrated in Examples 5, 7 and 11 and FIG. 17.

Very nonpolar analytes such as posaconazole can show inconsistentrecovery with some solvents and pH conditions tested (see Example 9).One skilled in the art will recognize that the solvent, sorbent, solventstrength and pH can be optimized for recovery of very nonpolar analytes,while possibly sacrificing removal of certain matrix interfering agents.Similarly, removal of all matrix interfering agents can be achieved toproduce a cleaner sample while possibly sacrificing recovery of allanalytes. Depending on the analytical objectives, compromising removalof all matrix constituents can be acceptable if on balance, the analytecan be quantitated more reliably. Similarly, compromising analyterecovery can be desirable if on balance, more accurate quantitation ispossible having achieved a significantly cleaner sample.

IV. Methods for Preparing a Device for Reducing Matrix Effects inBioanalytical Samples.

In additional embodiments, methods are provided for preparing devicesfor reducing matrix effects in a bioanalytical sample. In oneembodiment, a method is provided for preparing a device for reducingmatrix effects in a bioanalytical sample, comprising the followingsteps: a) providing a support capable of containing a quantity ofsorbent and a filtering means; and b) providing an amount of sorbenteffective to retain matrix interfering agents present in the sample, anda filtering means for removing precipitated proteins present in thesample; and c) assembling the filtering means and the sorbent within thesupport. The method can further comprise providing a retaining means forthe sorbent to hold it in place in the support, and/or supporting meansfor the filtering means to hold it in place in the support. Inadditional embodiments, the support further comprises a reservoircapable of containing the bioanalytical sample and solvent added toprecipitate proteins. In a particular embodiment, a device is preparedfor reducing matrix effects in a bioanalytical sample comprising thefollowing steps: a) providing a support capable of supporting a sizeexclusion filter and capable of containing a quantity of sorbent and abioanalytical sample; b) assembling a size exclusion filter within thesupport; c) assembling the sorbent into the support on top of the sizeexclusion filter; and d) optionally providing a retaining means for thesorbent to hold it in place in the support.

The following devices and method for preparation are nonlimitingexamples of devices that can be prepared and utilized for reducingmatrix effects and precipitated proteins in bioanalytical samples.

1. A device for use in a multiwell format can be prepared comprising thefollowing steps: preparing a slurry of 10 mg/ml Polaris® C18-A (10 μm)in MeOH and adding 2 ml (20 mg) to each well of a Captiva® 96 well plateincluding a 0.2 μm size exclusion filter (polypropylene); removing theMeOH by suction through the filters, and placing a frit on top of eachsorbent bed. The device is then ready for use.

2. A device for use as an individual filter cartridge can be preparedcomprising the following steps: providing an individual filter cartridgewith a frit for supporting a size exclusion membrane, assembling thefilter onto the frit, and further assembling a second frit on top of themembrane to secure it, applying sorbent on top of the second frit as aslurry in MeOH (20 mg from a 10 mg/ml slurry), removing the MeOH, andapplying a third frit to the top of the sorbent to secure the sorbent.

3. A device for inline use can be prepared comprising the followingsteps: providing a column body of metal or PEEK, inserting a frit in oneend followed by the filter means, followed optionally by an additionalsecuring frit; sorbent is inserted as a slurry or dry powder, followedby an additional securing means, capped off using hardware with anappropriate outlet and inlet to interface for use with a column.Alternatively, a sol gel monolith can be prepared in a column body,dried, calcinated and modified to provide a desired bonded phase, andequipped with the necessary hardware for inline use.

4. A device for use in a pipette tip format can be prepared comprisingthe following steps: providing a pipette tip and inserting a filtermeans into the small opening (the tip) of the pipette tip or by forminga monolithic filter means directly in the pipette tip by in situpolymerization, followed by assembling a plug of glass fiber monolithicsorbent prepared as described in U.S. Patent Application Publication No.20060216206 to Hudson or alternatively by providing a quantity ofparticulate or monolithic sorbent into the pipette tip adjacent to thefilter means. Pipette tips can also be utilized for reducing matrixeffects without utilizing filter means if protein precipitates are notpresent in the sample, in which case the pipette tips can be prepared asdescribed in U.S. Patent Application Publication No. 20060216206, suchas Omix IQe Tomtec tips (Varian, Inc., Palo Alto, Calif.).

5. A device for use as a luer based syringe filter can be prepared in amanner similar to that utilized to prepare a 96 well plate. A luerdevice can be fitted with a filter means and sorbent of desiredselectivity and pore size. For example, the filtering means can be aCaptiva® or Millipore filter 0.2 μm pore size and the sorbent can be aSpec® disk (available from Varian, Inc. Palo Alto, Calif.), or aplurality of Spec® disks or a particulate sorbent. The order of thesorbent and filtering means is not important, i.e., the sorbent or thefilter can be placed at the female or male end of the device However, itwould be conventional to filter out particulates prior to contacting thesample with the sorbent.

The devices can also be prepared by pouring or placing dry sorbentparticles or monolithic sorbents (e.g., a functionalized monolithicsorbent comprising a glass fiber matrix embedded with a bonded phase, ora sol gel monolith that is modified to produce a bonded phase, orpolymer modified porous substrates, etc.) on top of the filter and/orfrit or polymerizing polymer based monolithic sorbents (e.g., polymericsorbents comprising polar functionalities such as amide functionality)on top of the filter and/or frit.

One skilled in the art will be able to practice and assemble these othersimilar devices, understanding that the order of contact of thebioanalytical sample with sorbent and filtering means is generally notimportant. In addition, the devices can include reservoirs for preparingthe protein precipitates directly in the device, followed by adsorptionand filtration, all in one step, as demonstrated in Example 11. It ispreferred that the protein precipitation be performed prior to allowingthe sample contact with the sorbent, as protein adsorption couldadversely affect the ability of the sorbent to retain matrix interferingagents. However, this is not a necessary condition for use, and oneskilled in the art can vary the procedures to determine if it ispreferable to perform the precipitation prior to contacting the samplewith the sorbent.

One skilled in the art will further be able to substitute similarmaterials to provide devices having slightly different attributes. Forexample, any filtering means can be utilized, such as filters fromMillipore, Porex, Advantec, and the like, so long as the pore sizeutilized is appropriate for the application. Similarly, filters ofpolypropylene, PVDF, PTFE, nitrocellulose, etc., can be utilized, asdesired for particular applications. Reversed phase sorbents lackingpolar modification can be appropriate when analytes of interest arerelatively polar (e.g., log P<2) and are not well retained under thesolvent conditions associated with precipitated protein solutions (>50%(v/v) organic solvent).

It is also generally preferred that the device provide forunidirectional flow, that is, that the device have an inlet and outletside, and the sample is applied to one surface or inlet of the deviceand the treated sample exit from the outlet or other side of the device,having passed through the sorbent and the filtering means in aunidirectional fashion. However, in certain embodiments, the filteringmeans can be placed at the entrance to exclude particles from enteringthe device (e.g., a pipette tip implementation) while the sorbent isplaced adjacent the filtering means such that the protein precipitatesare filtered out of the sample as they enter the device and then thesample contacts the sorbent and afterwards is ejected back through thefiltering means.

In additional embodiments, the sorbent and filtering means are integralwith one another, for example, as where the filtering means is amonolithic inorganic substrate (e.g., a sol gel monolith) havingmacropores of the desired size for excluding precipitated proteinparticles and is surface modified to produce a reversed or polarmodified reversed bonded phase for selectively adsorbing matrixinterfering agents from the sample.

V. Applications and Methods of Use

The articles and methods of the present invention can be advantageouslyused in the preparation of samples for chromatographic and analyticalseparations applications, where the cleaner sample lacking protein andlipid/surfactant contaminants, for example, does not contaminatechromatographic media and instrumentation, which results in significantdown time for cleaning and maintenance, instrument drift andinconsistencies, and even effects on sorbent selectivity and analyteretention times.

The devices can be utilized in conjunction with capillary columns,cartridge systems, or conventional HPLC systems, as well asmicrofluidics applications. The devices and methods are particularlyadvantageously applied in the preparation of samples for high throughputscreening of plasma analytes (or other protein containing solutions)with mass spectrometric detection of analytes, where the reduction inmatrix interfering agents and precipitated proteins performed in asingle quick step provides extraordinary convenience and ease of use forthe busy laboratory worker. The devices can also be utilized in SPE.

VI. Advantages of the Invention

The inventive combination device comprising protein precipitation filterand a sorbent to remove lipids, surfactants and other matrix interferingagents allows the user to process the sample in a manner similar to thatused in a typical protein precipitation/filtration procedure butprovides the added benefit of reducing matrix effects as well asremoving proteins in one step. Use of the device dramatically simplifiesthe process of preparing clean bioanalytical samples prior to analysis.A separate sample treatment step utilizing SPE is not necessary.Therefore, use of the combination device provides much more efficientsample preparation, decreases labor and cost and time required forsample analysis.

An exemplary embodiment is described in Examples 10 and 11, anddemonstrates the dramatic improvement in sample preparation procedurespossible using the devices of the present invention. As described inExample 11, the sample could be subjected to both protein precipitationprocedures and stripped of matrix interfering agents (in this case,surfactants and phospholipids) in a single step using a single deviceand a pipetter. Overall, sample preparation required only a few minutesof time to measure and mix samples plus solvents, then a few seconds torecover the treated samples now ready for immediate analysis.

Accordingly, some advantages and characteristics of the present articlesand methods include: ease of use, convenience, one stepprotein/lipid/surfactant stripping is possible; saving time, solvents,materials, labor and cost.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessindicated otherwise, temperature is in degrees ° C. and pressure is ator near atmospheric. All solvents were purchased as HPLC grade, and allreactions were routinely conducted in the air unless otherwiseindicated.

Abbreviations:

ACN Acetonitrile

LC/MS-MS Liquid chromatography-mass spectrometry/mass spectrometry

SDS Sodium dodecyl sulfate

EXAMPLE 1 Phospholipid Build Up on a Chromatography Column

Dilute porcine plasma was injected into a polar modified C18-A HPLCcolumn (40 mm×4.0 mm), and plasma constituents were eluted using agradient program of ACN and 0.1% formic acid ramping from 10% to 90% ACNin 1 min after a 40 second hold. High organic elution was held for 40seconds before returning to 10% to re-equilibrate the column (see Table13).

The results are shown in FIG. 2, which demonstrates that thephospholipids (phosphatidylcholine) gradually accumulate on the columnover the course of 50 injections. LC-MS/MS traces are shown withdetection of the phosphatidylcholine eluition at m/z 184.0→184.0according to the method described by Little (2006) J. Chromatog. 833,219).

EXAMPLE 2 Correlation of Analyte Detection with Phosphatidylcholine andTween 80 Removal by Various Sorbents

A comparison of the abilities of various sorbents to removephospholipids and the surfactant Tween 80 was performed. Porcine plasmasamples (0.2 ml) were treated to precipitate proteins by the addition ofeither acetonitrile (ACN) or 1.0% formic acid in ACN (0.6 ml), thencentrifuged to remove precipitated proteins (Sorvall 50 ml rotor 5000rpm 20 min). The supernatants were removed and spiked with the followingpharmaceutical analytes: zolpidem, warfarin, quinidine, sulindac,loratadine, loperamide and Tween 80 (5 mg/ml). The spiked plasma sampleswere then filtered through a test sorbent: either ND 06262, ND 06265, ND06267 (instrAction GmbH, Ludwigshafen, DE), or Polaris® C18-Amide(Varian, Inc., Palo Alto, Calif.) 10 μm silica beads. Sorbents wereassembled on top of a Captiva® 0.45 μm membrane into a collection plate(10 mg or 20 mg sorbents). Ten μl aliquots were analyzed by HPLC using aVarian Polaris® C18-A column (3 μm particle diameter (50 mm×2.0 mm) witha gradient of A: 0.1% formic acid and B: acetonitrile according to Table1 using a Varian 1200L LC/MS-MS system.

TABLE 1 HPLC Elution Program Time % B Flow rate (μl/min)  0.00 10 350 1.00 10 350  8:00 90 350 53:00 90 350 53:01 10 350 55:00 10 350

Pharmaceutical analytes, phosphatidylcholine and Tween 80 MS/MSresponses were determined for 10 mg or 20 mg sorbent relative totreatment with the filtering membrane alone (control). Quantitation ionswere detected by their mass spectrometry transitions as shown in Table2.

TABLE 2 Analyte molecular ions Analytes MS transition Collision Energy(V) Zolpidem 308.1 → 235.0 −28 Warfarin 309.0 → 163.0 −11 Quinidine325.1 → 160.0 −21 Sulindac 357.1 → 233.0 −41 Loratadine 383.0 → 337.0−22.5 Loperamide 477.1 → 266.0 −22 Tween 80 309.0 → 309.0 −5Phosphatidylcholines 184.0 → 184.0 −5

The results are shown in FIGS. 3 through 10, and Tables 3-6 below. FIGS.3, 5, 7 and 9 demonstrate the reduction of phospholipids by treatmentwith 10 and 20 mg sorbents ND 06262, ND 06265, ND 06267, and Polaris®C18-Amide, respectively, when treated with 3:1 ACN precipitatedsupernatants with or without 1% formic acid. FIGS. 4, 6, and 8 showrelatively little reduction for Tween 80, however, FIG. 10 demonstratesmore significant removal of Tween 80 by Polaris® C18 Amide. Theseresults are presented in tabular form below.

TABLE 3 Results with ND 06262 10 mg sorbent 20 mg sorbent 3:1 3:1 ACN +1% 3:1 3:1 ACN + 1% ACN formic acid ACN formic acid Analyte Detectionvs. Captiva ® alone Zolpidem 99%  99% 150% 142% Warfarin 111%  129%  95%115% Quinidine — — — — Sulindac 105%  125% 116% 142% Loratadine 88% 113%122% 144% Loperamide 84% 111% 114% 138% Removal vs. Captiva ® aloneTween 80 21%  15%  11%  15% Phosphatidylcholines 40%  45%  72%  70%

TABLE 4 Results with ND 06265 10 mg sorbent 20 mg sorbent 3:1 3:1 ACN +1% 3:1 3:1 ACN + 1% ACN formic acid ACN formic acid Analyte Detectionvs. Captiva ® alone Zolpidem 109% 108% 110% 109% Warfarin 112% 106% 110%108% Quinidine 116% 108% 117% — Sulindac 137% 110% 114% 107% Loratadine117% 110% 113% 109% Loperamide 126% 116% 119% 121% Removal vs. Captiva ®alone Tween 80  1%  4%  −2%  −5% Phosphatidylcholines  14%  27%  61% 57%

TABLE 5 Results with ND 06267 10 mg sorbent 20 mg sorbent 3:1 3:1 ACN +1% 3:1 3:1 ACN + 1% ACN formic acid ACN formic acid Analyte Detectionvs. Captiva ® alone Zolpidem 87% 98% 92% 120% Warfarin 94% 96% 84% 122%Quinidine 98% 105%  — 146% Sulindac 94% 92% 120%  131% Loratadine 85%86% 98% 181% Loperamide 73% 85% 77% 126% Removal vs. Captiva ® aloneTween 80  3% −13%   8% −2.6%  Phosphatidylcholines 28% 19% 54%  48%

TABLE 6 Results with Polaris ® C18-Amide 10 mg sorbent 20 mg sorbent 3:13:1 ACN + 1% 3:1 3:1 ACN + 1% ACN formic acid ACN formic acid AnalyteDetection vs. Captiva ® alone Zolpidem  97% 106% 94% 99% Warfarin 103%112% 96% 97% Quinidine 118%  89% 94% 97% Sulindac 104% 106% 105%  97%Loratadine 106% 106% 95% 99% Loperamide 118% 118% 102%  107%  Removalvs. Captiva ® alone Tween 80  25%  27% 42% 41% Phosphatidylcholines  53% 70% 68% 81%

More surfactant and phospholipids were removed when using the largeramount of sorbent, indicating that there is a positive correlationbetween the amount of sorbent used and the amount of Tween 80 andphosphatidylcholines removed. However, sensitivity to analytes was notsacrificed in this bed mass range. Generally, removal of phospholipidsand Tween 80 resulted in enhancement of analyte detection, which isconsistent with removal of matrix interfering agents which results inreduction in ion suppression.

EXAMPLE 3 Surfactant Removal from Protein Precipitated Samples

Porcine plasma samples were spiked with analytes (Zolpidem, Warfarin,Sulindac, Loratadine, Vardenafil) to 1 ppm, together with surfactants(Triton X 100, Tween 80, and Sodium dodecyl sulfate (SDS)) to aconcentration of 1 mg/mL. Samples (200 μL) were precipitated using oneof four treatments:

-   -   400 μL 3% formic acid in acetonitrile (2:1)    -   600 μL 3% formic acid in acetonitrile (3:1)    -   400 μL 3% formic acid in methanol (2:1)    -   600 μL 3% formic acid in methanol (3:1)        Samples were centrifuged at 14,000 rpm for 12 min in a        mini-centrifuge. The supernatant was extracted as described in        Example 2 using 20 mg Polaris® C 18-A sorbent assembled on top        of a Captiva® 0.45 μm membrane. Ten μl aliquots were analyzed by        HPLC as described in Example 2 and Table 7 as shown below.

TABLE 7 HPLC Elution Program Time % B Flow rate (μl/min)  0.00 5 350 1.00 5 350  8:00 90 350 53:00 90 350 53:01 5 350 55:00 5 350

Analytes were detected using the molecular ions shown in Table 8.

TABLE 8 Analyte molecular ions Analytes MS transition Collision Energy(V) Zolpidem 308.1 → 235.0 −28 Warfarin 309.0 → 163.0 −11 Sulindac 357.1→ 233.0 −41 Loratadine 383.0 → 337.0 −22.5 Vardenafil 489.2 → 151.0−35.5 Triton X-100 625.4 → 625.4 −16.5 Tween 80 309.0 → 309.0 −5Phosphatidylcholines 184.0 → 184.0 −5

The results are summarized in Table 9. Chromatographic results are shownin FIGS. 11-14. FIG. 11 illustrates three chromatograms each of plasmaprotein precipitated using 2:1 MeOH (A-F) or 2:1 ACN (G-L) with 3%formic acid in both solutions; i.e., each chromatogram was performed intriplicate. FIG. 11A-C illustrate chromatograms obtained for proteinprecipitated plasma untreated with sorbent, while FIG. 11D-F illustratethe effect of treatment with 20 mg Polaris® C 18-A (the reduction inTween 80 in the treated sample). Similarly, FIG. 11G-I illustratechromatograms obtained for protein precipitated plasma untreated withsorbent, while FIG. 11J-L illustrate the effect of treatment with 20 mgPolaris® C18-A (the reduction in Tween 80 in the treated sample).

FIG. 12 illustrates three chromatograms each of plasma proteinprecipitated using 2:1 MeOH (A-F) or 2:1 ACN (G-L) with 3% formic acidin both solutions; i.e., each chromatogram was performed in triplicate.FIG. 12A-C illustrate chromatograms obtained for protein precipitatedplasma untreated with sorbent, while FIG. 12D-F illustrate the effect oftreatment with 20 mg Polaris® C 18-A (the reduction in Triton X-100 inthe treated sample). Similarly, FIG. 12G-I illustrate chromatogramsobtained for protein precipitated plasma untreated with sorbent, whileFIG. 12J-L illustrate the effect of treatment with 20 mg Polaris® C18-A(the reduction in Triton X-100 in the treated sample).

FIG. 13 illustrates three chromatograms each of plasma proteinprecipitated using 2:1 MeOH (A-F) or 2:1 ACN (G-L) with 3% formic acidin both solutions; i.e., each chromatogram was performed in triplicate.FIG. 13A-C illustrate chromatograms obtained for protein precipitatedplasma untreated with sorbent, while FIG. 13D-F illustrate the effect oftreatment with 20 mg Polaris® C18-A (the reduction in SDS in the treatedsample). Similarly, FIG. 13G-I illustrate chromatograms obtained forprotein precipitated plasma untreated with sorbent, while FIG. 13J-Lillustrate the effect of treatment with 20 mg Polaris® C18-A (thereduction in SDS in the treated sample).

FIG. 14 illustrates three chromatograms each of plasma proteinprecipitated using 2:1 MeOH (A-F) or 2:1 ACN (G-L) with 3% formic acidin both solutions; i.e., each chromatogram was performed in triplicate.FIG. 14A-C illustrate chromatograms obtained for protein precipitatedplasma untreated with sorbent, while FIG. 14D-F illustrate the effect oftreatment with 20 mg Polaris® C18-A (the reduction inphosphatidylcholines in the treated sample). Similarly, FIG. 14G-Iillustrate chromatograms obtained for protein precipitated plasmauntreated with sorbent, while FIG. 14J-L illustrate the effect oftreatment with 20 mg Polaris® C18-A (the reduction inphosphatidylcholines in the treated sample).

TABLE 9 Results (n = 3) Surfactant removal 2:1 3:1 2:1 3:1 with sorbentmethanol + methanol + ACN + ACN + relative to 3% formic 3% formic 3%formic 3% formic filtration alone acid acid acid acid Tween 80 90% 91%76% 70% Triton X 100 −10%  −0.4%  32% 20% SDS 85% 40%  6%  6% Phosphati-99% 99% 84% 91% dylcholines

As shown in FIGS. 11-14, and Table 9, surfactants and lipids could besubstantially removed from the protein precipitated samples using one ormore of the protocols tested. For example, use of MeOH at either 2:1 or3:1 dilution of plasma with 3% formic acid showed substantially completeremoval of phosphatidylcholines and Tween 80 from samples. Use of ACN ateither 2:1 or 3:1 dilution of plasma with 3% formic acid showedsubstantially complete removal of phosphatidylcholines and significantremoval of Tween 80 from samples. Removal of SDS was more dependent onstrength of the eluting solvent, as 85% of SDS could be removed with 2:1MeOH, while only 40% could be removed with 3:1 MeOH. Using Polaris C18-Aas the sorbent, ACN was not effective to remove SDS from samples.Removal of Triton X-100 was less effective under these conditions, withsignificant removal using ACN but not with MeOH.

EXAMPLE 4 Effect of Acid Concentration on Lipid Removal

Ten mL porcine plasma samples were spiked with analytes (Zolpidem,Warfarin, Sulindac, Loratadine, Loperamide, Vardenafil) to 1 ppm,together with 5 mg/mL Tween 80. Samples were precipitated by using 30 mLof one of three treatments:

-   -   Acetonitrile    -   1% formic acid in acetonitrile    -   2% formic acid in acetonitrile        Samples were centrifuged at 8,000 rpm for 30 min at 15 C using a        Sorvall 50 ml rotor. Samples of supernatant (800 μL) were        filtered as in Example 2 using 20 mg of ND 06262 or Polaris®        C18-Amide sorbent assembled on top of a Captiva® 0.45 μm        membrane into a collection plate under full vacuum.

Ten μl aliquots were analyzed by HPLC as described, and analytes weredetected by their mass spectrometric transitions, as described inExample 3. The results are summarized in Tables 10 and 11.Chromatographic results are shown in FIGS. 15 and 16. FIG. 15illustrates chromatograms generated using untreated samples (A, C, E)and samples treated with 20 mg Polaris® C18-Amide sorbent (B, D, F).Treatment of samples with 3:1 ACN without pH modifier is shown in FIGS.15A and B, illustrating the reduction in phosphatidylcholines in thesample. Treatment of samples with 3:1 ACN with 1% formic acid is shownin FIGS. 15C and D, illustrating the reduction in phosphatidylcholinesin the sample. Treatment of samples with 3:1 ACN with 2% formic acid isshown in FIGS. 15E and F, illustrating the reduction inphosphatidylcholines in the sample.

TABLE 10 Analyte detection using Polaris C18 Amide sorbent to removematrix interfering agents 3:1 ACN + 1% 3:1 ACN + 2% 3:1 ACN formic acidformic acid Recovery vs. Captiva ® filter alone Zolpidem 100.3% 106.1%189.3% Warfarin 118.0% 112.1% 107.9% Sulindac 117.5% 110.1% 114.0%Loratadine 114.9%  99.9%  99.4% Loperamide 116.6% 107.6% 117.1%Vardenafil 116.0%  93.3% 120.0% Removal vs. Captiva ® filter alone Tween80   54%   48%   37% Phosphatidylcholine   76%   86%   95%

These results demonstrate a marked relationship betweenphosphatidylcholine removal and acid concentration with Polaris® C18Amide, such that 2% formic acid resulted in the especially enhanceddetection of Zolpidem. At this highest acid concentration tested and inACN, nearly complete removal of phosphatidylcholine was achieved, evenfor lysophosphatidylcholine, which is notably difficult to remove, andpossibly resulting in greater enhancement in analyte detection. Tween 80removal was also significant. Removal of these matrix interfering agentsresults in analyte dependent enhancement of signal, due to the variableeffects of these ion suppression agents on individual analyte signals.

TABLE 11 Analyte detection using ND06262 sorbent to remove matrixinterfering agents 3:1 ACN + 1% 3:1 ACN + 2% 3:1 ACN formic acid formicacid Recovery vs. Captiva ® filter alone Zolpidem 88.8% 108.8%  101.2% Warfarin 113.1%  97.5% 103.8%  Sulindac 103.7%  93.0% 97.8% Loratadine100.0%  87.6% 90.5% Loperamide 96.5% 90.7% 98.2% Vardenafil 88.8% 79.0%84.9% Removal vs. Captiva ® filter alone Tween 80   6%  −8%  −18%Phosphatidylcholine  26%  58%  53%

ND06262 also demonstrates significant removal of phosphatidylcholine,however, under these solvent conditions, this sorbent demonstrateslittle efficacy in removing Tween 80 from the plasma samples. FIG. 16illustrates chromatograms generated using untreated samples (A, C, E)and samples treated with 20 mg ND06262 sorbent (B, D, F). Treatment ofsamples with 3:1 ACN without pH modifier is shown in FIGS. 16A and B,illustrating the reduction in phosphatidylcholines in the sample.Treatment of samples with 3:1 ACN with 1% formic acid is shown in FIGS.16C and D, illustrating the reduction in phosphatidylcholines in thesample. Treatment of samples with 3:1 ACN with 2% formic acid is shownin FIGS. 16E and F, illustrating the reduction in phosphatidylcholinesin the sample.

EXAMPLE 5 Methanol Testing

Porcine plasma samples (100 μL) were spiked with analytes (Zolpidem,Warfarin, Sulindac, Loratadine, Loperamide, Vardenafil) to 1 ppm,together with Tween 80 at 5 mg/mL. Samples were precipitated by dilutingplasma samples with 200 μL of methanol/3.0% formic acid. Samples werecentrifuged at 14,000 rpm for 15 min at room temperature in amicrocentrifuge. Samples of supernatant were filtered as in Example 2using 20 mg of Polaris® C18-A sorbent or simply transferred directly toa collection plate.

Ten μl aliquots were analyzed by HPLC as described in Example 3. Theresults are summarized in Table 12. Chromatographic results are shown inFIG. 17, pointing out particular species of phosphatidylcholinesobserved. FIG. 17A-C illustrate three chromatograms of proteinprecipitated plasma untreated with sorbent (the experiment was performedin triplicate). FIG. 17D-F illustrate three chromatograms of proteinprecipitated plasma treated with 20 mg of Polaris® C 18-A sorbentassembled on top of a Captiva® 0.45 μm membrane (the experiment wasperformed in triplicate).

TABLE 12 Analyte detection and removal of matrix interfering agentsRecovery vs. precip. only Zolpidem 109.6% Warfarin 100.5% Sulindac136.7% Loratadine 103.4% Loperamide 106.7% Vardenafil 106.5% Removal vs.precip. only Tween 80 81.8% Phosphatidylcholine 97.3%

These results demonstrate the nearly complete removal of both Tween 80and phospholipids present in the samples and complete recovery ofanalytes when using the Polaris® C18-A sorbent under these solventconditions. This combination of sorbent and protein precipitationconditions results in the surprisingly complete removal of matrixinterfering agents with no loss of analytes in a simpleprecipitation/extraction procedure.

EXAMPLE 6 Sorbent Selectivity Testing

Porcine plasma was protein precipitated using a 3:1 ACN dilution, andproteins were removed by centrifugation as described in Example 4.Samples of the plasma were spiked with the following analytes:Amitriptyline, Sumatriptan, Lamotrigine, Loratadine, Clozapine, andQuetiapine to 10 ppm, together with Tween 80 at 5 mg/mL. Ten μl aliquotsof supernatant, analytes and Tween 80 were analyzed by HPLC using avarious sorbents (40 mm×4.0 mm) with a gradient of A: 0.1% formic acidand B: acetonitrile according to Table 13 using a Varian 1200L LC/MS-MSsystem. The following sorbents were tested: Polaris® C18, Polaris®C18-A, and Focus® (Varian, Inc., Palo Alto, Calif.) and ND 06047(instrAction GmbH, Ludwigshafen, DE).

TABLE 13 HPLC Elution Program Time % B Flow rate (μl/min) 0.00 10 5000.66 10 500 1.66 90 500 2.33 90 500 2.34 10 500 3.00 10 500

The quantitation of the specific analytes and matrix interfering agentswere determined using the mass spectrometric transitions shown in Table12.

TABLE 14 Analyte molecular ions Figure Collision designation Analytes MStransition Energy A Phosphatidylcholines 184.0 → 184.0 −5 B Tween 80309.0 → 309.0 −5 C Lamotrigine 256.0 → 256.0 −4 D Amitriptyline 278.1 →233.0 −15 E Sumatriptan 296.0 → 58.0 −13 F Clozapine 327.0 → 270.0 −21 GLoratadine 383.0 → 337.0 −22.5 H Quetiapine 384.0 → 309.2 −5

The results are shown in FIGS. 18-21, with A-H indicating analytetracings as shown in Table 14. FIG. 18 shows the elution of analytes andmatrix interfering agents on a pure reversed phase sorbent, C-18modified silica (Polaris® C18), and demonstrates that there is someoverlap in retention times for some of the later eluting analytes withthe matrix interfering agents phosphatidylcholines and Tween 80 due tothe strong retention of nonpolar analytes on this sorbent. Thecalculated selectivity between Loratadine (the least polar analyte, logP=3.65) and the leading edge of phosphatidylcholines or Tween 80 peaksis about 1.

FIG. 19 shows the elution of analytes and matrix interfering agents on apolar modified reversed phase sorbent, Polaris® C18-A, and demonstratesthat there is very little overlap in retention times for the latereluting analytes with the matrix interfering agents phosphatidylcholinesand Tween 80 due to the less strong retention of nonpolar analytes onthis sorbent in acidic solvent elution conditions (basic analytes areless retained by the polar modified sorbent.).

FIG. 20 shows the elution of analytes and matrix interfering agents on apolar modified reversed phase sorbent, Focus (a reversed phase amidemodified aromatic polymer), and demonstrates that there is very littleoverlap in retention times for some of the later eluting analytes withthe matrix interfering agents phosphatidylcholines and Tween 80 due tothe less strong retention of nonpolar analytes on this sorbent.

Finally, FIG. 21 shows the elution of analytes and matrix interferingagents on instrAction ND06047 (a polymer network formed on silicabeads), and demonstrates that there is very little overlap in retentiontimes for some of the later eluting analytes with the matrix interferingagents phosphatidylcholines and Tween 80 due to the less strongretention of nonpolar analytes on this sorbent. There is very littleresolution between certain of the analytes as well under these elutionconditions, but resolution between analytes is not intended for thesesorbents.

These chromatograms demonstrate the selectivity of the sorbents testedover a broad range of analyte and matrix interfering agent retentiontimes. The calculated selectivity between Loratadine (the least polaranalyte, log P=3.65) and the leading edge of phosphatidylcholines orTween 80 peaks is greater than 1 for all polar modified reversed phasesorbents, allowing the selective separation of even strongly nonpolaranalytes from the matrix interfering agents phosphatidylcholines andTween 80.

EXAMPLE 7 Particulate Size and Method of Protein Precipitation

Porcine plasma samples (0.2 ml) were protein precipitated using one ofthe following four treatments to yield either 66% (v/v) or 75% (v/v)organic solvent solutions:

-   -   addition of 0.4 ml MeOH with 3% formic acid    -   addition of 0.6 ml MeOH with 3% formic acid    -   addition of 0.4 ml ACN with 1% formic acid    -   addition of 0.6 ml ACN with 1% formic acid.

The protein precipitated samples were passed through a Captiva® plateusing 0.45 μm or 0.2 μm pore size filters or a Captiva® plate containingsorbent (20 mg 10 μm C18-A) using 0.45 μm or 0.2 μm pore size filters.Transmittance (% T, 1 cm path, relative to MeOH blank) was measured at524 nm to determine turbidity, as a measure of protein particulatesproduced by the different protein precipitation procedures and theability of the Captiva® filter plate with or without sorbent to removethe particulates. The results are shown in FIG. 22.

As shown in FIG. 22, protein precipitation using MeOH dilution producesthe smallest particulates, which could not be removed by filtrationthrough 0.45 μm pore size Captiva® filters alone (% T was zero). Theaddition of 20 mg sorbent removed a small amount of the proteinprecipitates (% T was ˜25%). Use of 0.2 μm pore size filters providedbetter results for 60% (v/v) MeOH precipitated samples (˜50% T wasachieved); however, % T was still zero for 66% (v/v) MeOH precipitatedsamples. Addition of 20 mg sorbent provided better particulate removal,with ˜20% T for 66% (v/v) MeOH precipitated plasma, and ˜100% T for 75%(v/v) MeOH precipitated plasma.

Protein precipitation using ACN dilution produced larger particulatesthat could be partially removed by filtration through 0.45 μm pore sizeCaptiva® filters alone: % T was ˜5% for 66% (v/v) precipitation and ˜95%for 75% (v/v) precipitation. The addition of 20 mg sorbent removedslightly more of the protein precipitates: % T was ˜7% for 66% (v/v)precipitation and ˜98% for 75% (v/v) precipitation. Use of 0.2 μm poresize Captiva® filters alone provided maximal removal of particulates: %T was ˜97% for 66% (v/v) precipitation and ˜98% for 75% (v/v)precipitation. The addition of 20 mg sorbent removed slightly more ofthe protein precipitates: % T was ˜99% for 66% (v/v) precipitation and˜98% for 75% (v/v) precipitation.

Thus, the method of protein precipitation produces variable particulatesizes, which can be removed using an appropriate filtration methodpossessing the requisite particle size filtration capabilities.Acceptable protein particulate removal for 75% (v/v) MeOH precipitatedsamples was only achieved using 0.2 μm pore size Captiva® filters with20 mg sorbent. Acceptable protein particulate removal for 75% (v/v) ACNprecipitated samples was achieved using 0.45 μm pore size Captiva®filters with or without 20 mg sorbent or using 0.2 μm pore size Captiva®filters with or without sorbent; while for 66% (v/v) ACN, acceptableparticulate removal was achieved only with 0.2 μm pore size filters, andwas equivalent with and without sorbent.

EXAMPLE 8 Rate of Removal of Matrix Interfering Agents

Porcine plasma samples (0.2 ml) were protein precipitated by theaddition of 0.6 ml 1.0% formic acid in ACN. Protein precipitates wereremoved by centrifugation (5000 rpm for 20 minutes in a Sorvall 50 mlrotor). Samples were spiked with analytes (Zolpidem, Warfarin, Sulindac,Loratadine, Vardenafil) to 1 ppm and with Tween 80 to 5 mg/ml. Sampleswere contacted with the sorbents (20 mg Polaris® C18-Amide (10 μm) or ND06262) for the following time periods in a 96 well plate with a 0.45 μmCaptiva® filter and then separated from the sorbents by vacuumfiltration through a collection plate: approximately 10, 20, 50, 120seconds or 5, 10, 15, 60, 150 seconds. The contact time with sorbent wascontrolled by varying the flow rate by applying variable vacuum: −15inches Hg resulted in 10 seconds of contact with sorbent. Additionalapplied vacuum of −10, −5, −2.5, −2 inches of Hg gave the contact timesshown. Filtrates were then analyzed by HPLC as described in Example 2 todetermine the recovery of analytes and the amount of phosphatidylcholineand Tween 80 removed. The results are shown in FIGS. 23 and 24.

FIG. 23 demonstrates that approximately 75% of the phosphatidylcholinewas removed from the plasma samples by Polaris® C18 Amide within 10seconds of exposure to the sorbent, and did not change over the timeperiod tested, and that ND 06262 was able to remove at least 50% of thephosphatidylcholine within 5 seconds, and increased slightly to over 60%by 150 seconds of contact between the sample and the sorbent.

FIG. 24 demonstrates that approximately 40% of the Tween 80 was removedfrom the plasma samples by Polaris® C18 Amide within 10 seconds ofexposure to the sorbent, and this amount increased only slightly overthe time period tested. ND 06262 was able to remove very little of theTween 80 over the time period tested.

Analyte detection was dependent on contact time of the plasma sampleswith Polaris® C18 Amide. Analyte detection was greater than 100%(relative to filtration alone) for all contact times of 20 seconds orlonger, indicating that maximal phospholipids and Tween 80 removal hadoccurred by 20 seconds of exposure time to the sorbent. (data not shown)

Analyte detection was not as clearly dependent on contact time of theplasma samples with ND 06262, indicating that this sorbent was not aseffective at removing phosphatidylcholines and Tween 80 as Polaris® C18Amide under these solvent conditions. (data not shown)

EXAMPLE 9 Optimizing Solvent Conditions to Maximize Removal ofPhosphatidylcholines

Human plasma was spiked with the following analytes: pseudoephedrine,carbamazepine, desloratadine, propranolol, and posaconazole to 100 ng/ml(0.1 ppm). Aliquots of spiked plasma (0.1 ml) were removed (n=6) and 0.2ml or 0.3 ml organic solvent with or without pH modifier was added toeach aliquot to precipitate proteins. Denaturing solvents tested wereacetone, ACN, MeOH at 2:1 and 3:1 dilution of plasma volumes, with orwithout 2% formic acid. Each protein precipitated plasma sample waspassed through either a well of a Captiva® plate containing 20 mgPolaris® C18-A sorbent, or transferred to another 96 well plate. Water(0.2 ml) was added to dilute each sample prior to HPLC and 10 μlaliquots were analyzed using an API 5000 LC/MS-MS system. Recovery wascalculated by dividing the average area count of each analyte whichpassed through the plate by the average area count of each analyte whichdid not pass through the plate.

The results are shown in Tables 15-18 below.

TABLE 15 Solvent dilution 2:1 without pH modifier Analyte Log P AcetoneACN MeOH Recovery pseudoephedrine 1.51 136%  90% 88% carbamazepine 2.6988% 89% 90% desloratadine 3.23 76% 78% 57% propranolol 3.59 82% 82% 70%posaconazole 5.66 65% 67% 44% Removal lysoPC 17%  2% 94% PC 100%  100% 100% 

TABLE 16 Solvent dilution 2:1 with pH modifier (2% formic acid) AnalyteLog P Acetone ACN MeOH Recovery pseudoephedrine 1.51 98% 86% 93%carbamazepine 2.69 96% 84% 90% desloratadine 3.23 96% 69% 81%propranolol 3.59 96% 79% 87% posaconazole 5.66 82% 72% 37% RemovallysoPC  2%  5% 100%  PC 100%  100%  100% 

TABLE 17 Solvent dilution 3:1 without pH modifier Analyte Log P AcetoneACN MeOH Recovery pseudoephedrine 1.51 161% 93% 99% carbamazepine 2.69105% 94% 101%  desloratadine 3.23 100% 87% 80% propranolol 3.59 101% 88%90% posaconazole 5.66  95% 91% 71% Removal lysoPC  28%  5% 32% PC  97%100%  100% 

TABLE 18 Solvent dilution 3:1 with pH modifier (2% formic acid) AnalyteLog P Acetone ACN MeOH Recovery pseudoephedrine .51 100%  90% 109%carbamazepine 2.69 97% 89% 110% desloratadine 3.23 95% 79%  99%propranolol 3.59 98% 88% 108% posaconazole 5.66 124%  88%  66% RemovallysoPC 11% 10%  62% PC 97% 100%  100%

These results demonstrate the surprisingly complete removal ofphosphatidylcholines, including significant amount oflysophosphatidylcholines, while recovering analytes of a wide range ofpolarities from the sorbent.

EXAMPLE 10 Preparation of a Device for Reducing Matrix Effects

A device was prepared for reducing matrix effects in a bioanalyticalsample comprising the following steps: a slurry of 10 mg/ml Polaris®C18-A (10 μm) in MeOH was prepared and 2 ml (20 mg) was added to eachwell of a a Captiva® 96 well plate including a 0.2 μm size exclusionfilter (polypropylene). The MeOH was removed by suction through thefilters, and a frit was placed on top of each sorbent bed. The devicewas then ready for use.

EXAMPLE 11 Performance of a Device for Reducing Matrix Effects

Porcine plasma samples were spiked with analytes (Zolpidem, Warfarin,Sulindac, Loratadine, Loperamide, Vardenafil) to 1 ppm each and withTween 80 to 5 mg/ml and 0.2 ml aliquots were protein precipitated inwells of a modified 96 well Captiva® plate. The plate was modified tocontain 20 mg Polarise C18-A (10 μm) on top of the 0.2 μm polypropyleneCaptiva® filter. MeOH/3% formic acid (0.6 ml) was added to the sampleand mixed by pipetting (5 cycles of 0.6 ml) and then filtered throughthe plate by vacuum filtration. Three different lots of Polaris® C18-Asorbent were tested. Treated samples were analyzed using HPLC asdescribed in Example 3.

The results demonstrated >100% recovery of analytes on average,indicating that matrix effects had been reduced. Approximately 85% ofTween 80, 65% of lysophosphatidylcholines, and 99% ofphosphatidylcholines were removed using the device (data not shown).Overall, sample preparation required only a few minutes of time tomeasure and mix samples plus solvents, then a few seconds to recover thetreated samples, now ready for immediate analysis.

1-21. (canceled)
 22. A device for reducing matrix effects in a proteinprecipitated bioanalytical sample comprising: a) a support, b) a sorbentassociated with the support capable of binding matrix interfering agentspresent in the bioanalytical sample; and c) a filtering means forremoving precipitated protein particles, wherein the filtering means ischaracterized in having pore sizes between about 0.05 μm and about 0.5μm in diameter.
 23. The device of claim 22, wherein the filtering meansis selected from a size exclusion filter or a polymeric or inorganicmonolith having a maximum pore size less than or equal to the diameterof the particles to be removed from the sample.
 24. The device of claim22, wherein the filtering means is integral with the sorbent orassociated with the sorbent.
 25. The device of claim 22, wherein thefiltering means is a porous monolith having macropores of a diametersufficiently small so as to exclude particles from the sample, and thesorbent is a reversed phase or polar modified reversed phase bonded tothe porous monolith.
 26. The device of claim 22, wherein the sorbent ischaracterized by sufficient selectivity between the matrix interferingagents and analytes of interest to provide retention of the matrixinterfering agents while providing elution of the analytes of interest.27. The device of claim 26, wherein the sorbent is characterized by aselectivity greater than 1 between matrix interfering agents andanalytes of interest.
 28. The device of claim 22, wherein the sorbentcomprises a reversed phase or a polar modified reversed phase.
 29. Thedevice of claim 28, wherein the polar modified reversed phase is anamide modified reversed phase.
 30. The device of claim 22, wherein thematrix interfering agent is a surfactant, lipid, excipient, or dosingagent.
 31. The device of claim 22, adapted for use as a luer syringefilter, individual filter cartridge, a multiwall plate, pipette tip, oran inline column for multiple or single use.
 32. The device of claim 22,wherein the support further comprises reservoir means for performingprotein precipitation within the device.
 33. A method for reducingmatrix effects and removing protein precipitates in a bioanalyticalsample, said method comprising: a) providing the device of claim 22; b)contacting the bioanalytical sample with the sorbent; and c) elutinganalytes from the sorbent while retaining the matrix interfering agentsand precipitated proteins, wherein the amount of matrix interferingagents and proteins in the resulting treated sample is reduced.
 34. Themethod of claim 33, further comprising precipitating the proteins in thebioanalytical sample in the device prior to or simultaneously with thestep of contacting the bioanalytical sample with the sorbent.
 35. Themethod of claim 33, wherein step c) is performed using vacuum,centrifugal or electrokinetic force, gravity or capillary forces, orpositive pressure.
 36. The method of claim 33, wherein the matrixinterfering agents are surfactants, lipids, excipients, or dosingagents.
 37. The method of claim 33, wherein the sorbent is characterizedby sufficient selectivity between the matrix interfering agents andanalytes of interest to provide retention of the matrix interferingagents while providing elution of the analytes of interest.
 38. A methodfor reducing matrix effects and removing precipitated proteins in aprotein precipitated bioanalytical sample, the method comprising passingthe sample through the device of claim
 10. 39. A method for preparingthe device of claim 22 comprising the following steps: a) providing asupport capable of containing a quantity of sorbent and a filteringmeans; b) providing an amount of sorbent effective to retain matrixinterfering agents and filtering means effective to remove precipitatedproteins; and c) assembling the filtering means and the sorbent withinthe support.